90. Dhanda, AS; Yang, DN; Guttman, JA. (2021) Localization of alpha-actinin-4 during infections by actin remodeling bacteria.Anat. Rec. 304: 1400-1419 Localization of alpha-actinin-4 during infections by actin remodeling bacteria
diarrhea; E; coli; gut; listeriopod; listeriosis
Bacterial pathogens cause disease by subverting the structure and function of their target host cells. Several foodborne agents such as Listeria monocytogenes (L. monocytogenes), Shigella flexneri (S. flexneri), Salmonella enterica serovar Typhimurium (S. Typhimurium) and enteropathogenic Escherichia coli (EPEC) manipulate the host actin cytoskeleton to cause diarrheal (and systemic) infections. During infections, these invasive and adherent pathogens hijack the actin filaments of their host cells and rearrange them into discrete actin-rich structures that promote bacterial adhesion (via pedestals), invasion (via membrane ruffles and endocytic cups), intracellular motility (via comet/rocket tails) and/or intercellular dissemination (via membrane protrusions and invaginations). We have previously shown that actin-rich structures generated by L. monocytogenes contain the host actin cross-linker alpha-actinin-4. Here we set out to examine alpha-actinin-4 during other key steps of the L. monocytogenes infectious cycle as well as characterize the subcellular distribution of alpha-actinin-4 during infections with other model actin-hijacking bacterial pathogens (S. flexneri, S. Typhimurium and EPEC). Although alpha-actinin-4 is absent at sites of initial L. monocytogenes invasion, we show that it is a new component of the membrane invaginations formed during secondary infections of neighboring host cells. Importantly, we reveal that alpha-actinin-4 also localizes to the major actin-rich structures generated during cell culture infections with S. flexneri (comet/rocket tails and membrane protrusions), S. Typhimurium (membrane ruffles) and EPEC (pedestals). Taken together, these findings suggest that alpha-actinin-4 is a host factor that is exploited by an assortment of actin-hijacking bacterial pathogens. DOI PubMed
89. Dhanda, AS; Yang, DN; Kooner, A; Guttman, JA. (2021) Distribution of PDLIM1 at actin-rich structures generated by invasive and adherent bacterial pathogens.Anat. Rec. 304: 919-938 Distribution of PDLIM1 at actin-rich structures generated by invasive and adherent bacterial pathogens
CLP36; diarrhea; elfin; gut; intestine; listeriopod; PDZ‐ LIM
The enteric bacterial pathogens Listeria monocytogenes (Listeria) and enteropathogenic Escherichia coli (EPEC) remodel the eukaryotic actin cytoskeleton during their disease processes. Listeria generate slender actin-rich comet/rocket tails to move intracellularly, and later, finger-like membrane protrusions to spread amongst host cells. EPEC remain extracellular, but generate similar actin-rich membranous protrusions (termed pedestals) to move atop the host epithelia. These structures are crucial for disease as diarrheal (and systemic) infections are significantly abrogated during infections with mutant strains that are unable to generate the structures. The current repertoire of host components enriched within these structures is vast and diverse. In this protein catalog, we and others have found that host actin crosslinkers, such as palladin and alpha-actinin-1, are routinely exploited. To expand on this list, we set out to investigate the distribution of PDLIM1, a scaffolding protein and binding partner of palladin and alpha-actinin-1, during bacterial infections. We show that PDLIM1 localizes to the site of initial Listeria entry into cells. Following this, PDLIM1 localizes to actin filament clouds surrounding immotile bacteria, and then colocalizes with actin once the comet/rocket tails are generated. Unlike palladin or alpha-actinin-1, PDLIM1 is maintained within the actin-rich core of membrane protrusions. Conversely, alpha-actinin-1, but not PDLIM1 (or palladin), is enriched at the membrane invagination that internalizes the Listeria-containing membrane protrusion. We also show that PDLIM1 is a component of the EPEC pedestal core and that its recruitment is dependent on the bacterial effector Tir. Our findings highlight PDLIM1 as another protein present within pathogen-induced actin-rich structures. DOI PubMed
88. Dhanda, AS; Yu, C; Lulic, KT; Vogl, AW; Rausch, V; Yang, D; Nichols, BJ; Kim, SH; Polo, S; Hansen, CG; Guttman, JA. (2020) Listeria monocytogenes Exploits Host Caveolin for Cell-to-Cell Spreading.mBio 11 Listeria monocytogenes Exploits Host Caveolin for Cell-to-Cell Spreading
endocytosis; epsin-1; membrane protrusion; invaginations; actin; Listeria monocytogenes; actin-based motility; cell-to-cell spreading
Listeria monocytogenes moves from one cell to another using actin-rich membrane protrusions that propel the bacterium toward neighboring cells. Despite cholesterol being required for this transfer process, the precise host internalization mechanism remains elusive. Here, we show that caveolin endocytosis is key to this event as bacterial cell-to-cell transfer is severely impaired when cells are depleted of caveolin-1. Only a subset of additional caveolar components (cavin-2 and EHD2) are present at sites of bacterial transfer, and although clathrin and the clathrin-associated proteins Eps15 and AP2 are absent from the bacterial invaginations, efficient L. monocytogenes spreading requires the clathrin-interacting protein epsin-1. We also directly demonstrated that isolated L. monocytogenes membrane protrusions can trigger the recruitment of caveolar proteins in a neighboring cell. The engulfment of these bacterial and cytoskeletal structures through a caveolin-based mechanism demonstrates that the classical nanometer-scale theoretical size limit for this internalization pathway is exceeded by these bacterial pathogens. IMPORTANCE Listeria monocytogenes moves from one cell to another as it disseminates within tissues. This bacterial transfer process depends on the host actin cytoskeleton as the bacterium forms motile actin-rich membranous protrusions that propel the bacteria into neighboring cells, thus forming corresponding membrane invaginations. Here, we examine these membrane invaginations and demonstrate that caveolin-1-based endocytosis is crucial for efficient bacterial cell-to-cell spreading. We show that only a subset of caveolin-associated proteins (cavin-2 and EHD2) are involved in this process. Despite the absence of clathrin at the invaginations, the classical clathrin-associated protein epsin-1 is also required for efficient bacterial spreading. Using isolated L. monocytogenes protrusions added onto naive host cells, we demonstrate that actin-based propulsion is dispensable for caveolin-1 endocytosis as the presence of the protrusion/invagination interaction alone triggers caveolin-1 recruitment in the recipient cells. Finally, we provide a model of how this caveolin-1-based internalization event can exceed the theoretical size limit for this endocytic pathway. DOI PubMed
87. Aggarwal, P; Wei, LF; Cao, YP; Liu, Q; Guttman, JA; Wang, QY; Leung, KY. (2019) Edwardsiella induces microtubule-severing in host epithelial cells.Microbiol. Res. 229 Edwardsiella induces microtubule-severing in host epithelial cells
E. piscicida; Fish pathogen; Microtubule disassembly; Interaction with epithelial cells
Edwardsiella bacteria cause economic losses to a variety of commercially important fish globally. Human infections are rare and result in a gastroenteritis-like illness. Because these bacteria are evolutionarily related to other Enterobacteriaceae and the host cytoskeleton is a common target of enterics, we hypothesized that Edwardsiella may cause similar phenotypes. Here we use HeLa and Caco-2 infection models to show that microtubules are severed during the late infections. This microtubule alteration phenotype was not dependant on the type III or type VI secretion system (T3SS and T6SS) of the bacteria as Delta T3SS and Delta T6SS mutants of E. piscicida EIB202 and E. tarda ATCC15947 that lacks both also caused microtubule disassembly. Immunolocalization experiments showed the host katanin catalytic subunits A1 and A like 1 proteins at regions of microtubule severing, suggesting their involvement in the microtubule disassembly events. To identify bacterial components involved in this phenotype, we screened a 2,758 transposon library of E. piscicida EIB202 and found that 4 single mutations in the aipFHAGDC operon disrupted microtubule disassembly in HeLa cells. We then constructed three atp deletion mutants; they all could not disassemble host microtubules. This work provides the first clear evidence of host cytoskeletal alterations during Edwardsiella infections. DOI PubMed
86. Chua, MD; Bogdan, AC; Guttman, JA. (2019) Klebsiella pneumoniae Redistributes Katanin Severing Proteins and Alters Astral Microtubules during Mitosis.Anat. Rec.Klebsiella pneumoniae Redistributes Katanin Severing Proteins and Alters Astral Microtubules during Mitosis
Klebsiella pneumoniae; microtubules; katanins; mitosis
Klebsiella pneumoniae has become a growing concern within hospitals due to multidrug resistant strains and increasing mortality rates. Recently, we showed that at the subcellular level, K. pneumoniae compromises the integrity of the epithelia by disassembling the microtubule networks of cells through the actions of katanin microtubule severing proteins. In this study, we report on the observation that mitotic cells are targeted by K. pneumoniae and that during infections, the katanin proteins are excluded from the microtubule organizing centers of dividing cells, resulting in the alteration of the microtubule cytoskeleton. Anat Rec, 2019. (c) 2019 American Association for Anatomy DOI PubMed
85. Chua, MD; Liou, CH; Bogdan, AC; Law, HT; Yeh, KM; Lin, JC; Siu, LK; Guttman, JA. (2019) Klebsiella pneumoniae disassembles host microtubules in lung epithelial cells.Cell Microbiol. 21 Klebsiella pneumoniae disassembles host microtubules in lung epithelial cells
Klebsiella pneumoniae raises significant concerns to the health care industry as these microbes are the source of widespread contamination of medical equipment, cause pneumonia as well as other multiorgan metastatic infections and have gained multidrug resistance. Despite soaring mortality rates, the host cell alterations occurring during these infections remain poorly understood. Here, we show that during in vitro and in vivo K. pneumoniae infections of lung epithelia, microtubules are severed and then eliminated. This destruction does not require direct association of K. pneumoniae with the host cells, as microtubules are disassembled in cells that are distant from the infecting bacteria. This microtubule dismantling is dependent on the K. pneumoniae (Kp) gene ytfL as non-pathogenic Escherichia coli expressing Kp ytfL disassemble microtubules in the absence of K. pneumoniae itself. Our data points to the host katanin catalytic subunit A like 1 protein (KATNAL1) and the katanin regulatory subunit B1 protein (KATNB1) as the gatekeepers to the microtubule severing event as both proteins localise specifically to microtubule cut sites. Infected cells that had either of these proteins knocked out maintained intact microtubules. Taken together, we have identified a novel mechanism that a bacterial pathogen has exploited to cause microtubule destruction within the host epithelia. DOI PubMed
84. Dhanda, AS; Lulic, KT; Vogl, AW; Mc Gee, MM; Chiu, RH; Guttman, JA. (2019) Listeria Membrane Protrusion Collapse: Requirement of Cyclophilin A for Listeria Cell-to-Cell Spreading.J. Infect. Dis. 219 Listeria Membrane Protrusion Collapse: Requirement of Cyclophilin A for Listeria Cell-to-Cell Spreading
cell-to-cell spread; cyclophilin A; Listeria; Listeria monocytogenes
Background. Listeria generate actin-rich tubular protrusions at the plasma membrane that propel the bacteria into neighboring cells. The precise molecular mechanisms governing the formation of these protrusions remain poorly defined. Methods. In this study, we demonstrate that the prolyl cis-trans isomerase (PPIase) cyclophilin A (CypA) is hijacked by Listeria at membrane protrusions used for cell-to-cell spreading. Results. Cyclophilin A localizes within the F-actin of these structures and is crucial for their proper formation, as cells depleted of CypA have extended actin-rich structures that are misshaped and are collapsed due to changes within the F-actin network. The lack of structural integrity within the Listeria membrane protrusions hampers the microbes from spreading from CypA null cells. Conclusions. Our results demonstrate a crucial role for CypA during Listeria infections. DOI PubMed
83. Dhanda, AS; Lulic, KT; Yu, C; Chiu, RH; Bukrinsky, M; Guttman, JA. (2019) Listeria monocytogenes hijacks CD147 to ensure proper membrane protrusion formation and efficient bacterial dissemination.Cell. Mol. Life Sci. 76: 4165-4178 Listeria monocytogenes hijacks CD147 to ensure proper membrane protrusion formation and efficient bacterial dissemination
Comet/rocket tails; Cell-to-cell spreading; Actin-based motility; Infectious disease; Microbiology; Actin
Efficient cell-to-cell transfer of Listeria monocytogenes (L. monocytogenes) requires the proper formation of actin-rich membrane protrusions. To date, only the host proteins ezrin, the binding partner of ezrin, CD44, as well as cyclophilin A (CypA) have been identified as crucial components for L. monocytogenes membrane protrusion stabilization and, thus, efficient cell-to-cell movement of the microbes. Here, we examine the classical binding partner of CypA, CD147, and find that this membrane protein is also hijacked by the bacteria for their cellular dissemination. CD147 is enriched at the plasma membrane surrounding the membrane protrusions as well as the resulting invaginations generated in neighboring cells. In cells depleted of CD147, these actin-rich structures appear similar to those generated in CypA depleted cells as they are significantly shorter and more contorted as compared to their straighter counterparts formed in wild-type control cells. The presence of malformed membrane protrusions hampers the ability of L. monocytogenes to efficiently disseminate from CD147-depleted cells. Our findings uncover another important host protein needed for L. monocytogenes membrane protrusion formation and efficient microbial dissemination. DOI PubMed
82. Dhanda, AS; Yu, C; Guttman, JA. (2019) Distribution of CD147 During Enteropathogenic Escherichia coli and Salmonella enterica Serovar Typhimurium Infections.Anat. Rec. 302: 2224-2232 Distribution of CD147 During Enteropathogenic Escherichia coli and Salmonella enterica Serovar Typhimurium Infections
membrane ruffles; basigin; EMMPRIN
Enteropathogenic Escherichia coli (EPEC) and Salmonella enterica serovar Typhimurium (S. Typhimurium) are highly infectious gastrointestinal human pathogens. These microbes inject bacterial-derived effector proteins directly into the host cell cytosol as part of their disease processes. A common host subcellular target of these pathogens is the actin cytoskeleton, which is commandeered by the bacteria and is used during their attachment onto (EPEC) or invasion into (S. Typhimurium) the host cells. We previously demonstrated that the host enzyme cyclophilin A (CypA) is recruited to the actin-rich regions of EPEC pedestals and S. Typhimurium membrane ruffles. To further expand the growing catalogue of host proteins usurped by actin-hijacking bacteria, we examined the host plasma membrane protein and cognate receptor of CypA, CD147, during EPEC and S. Typhimurium infections. Here, we show that CD147 is enriched at the basolateral regions of pedestals but, unlike CypA, it is absent from their actin-rich core. We show that the CD147 recruitment to these areas requires EPEC pedestal formation and not solely bacteria-host cell contact. Additionally, we demonstrate that the depletion of CD147 by siRNA does not alter the formation of pedestals. Finally, we show that CD147 is also a component of actin-rich membrane ruffles generated during S. Typhimurium invasion of host cells. Collectively, our findings establish CD147 as another host component present at dynamic actin-rich structures formed during bacterial infections. (C) 2019 American Association for Anatomy. DOI PubMed
81. Chua, MD; Hipolito, KJ; Singerr, OB; Solway, J; Guttman, JA. (2018) SM22 is required for the maintenance of actin-rich structures generated during bacterial infections.Exp. Cell Res. 369 SM22 is required for the maintenance of actin-rich structures generated during bacterial infections
SM22; Transgelin; Enteropathogenic Escherichia coli; Listeria monocytogenes
The host actin cytoskeleton is utilized by an assortment of pathogenic bacteria to colonize and cause disease in their hosts. Two prominently studied actin-hijacking bacteria are enteropathogenic Escherichia coli (EPEC) and Listeria monocytogenes. EPEC form actin-rich pedestals atop its host cells to move across the intestinal epithelia, while Listeria monocytogenes generate branched actin networks arranged as actin clouds around the bacteria and as comet tails for propulsion within and amongst their host cells. Previous mass spectrometry analysis revealed that a member of the calponin family of actin-bundling proteins, transgelin/SM22 was enriched in EPEC pedestals. To validate that finding and examine the role of SM22 during infections, we initially immunolocalized SM22 in EPEC and L. monocytogenes infected cells, used siRNA to deplete SM22 and EGFP-SM22 to overexpress SM22, then quantified the alterations to the bacterially generated actin structures. SM22 concentrated at all bacterially-generated actin structures. Depletion of SM22 resulted in fewer pedestals and comet tails and caused comet tails to shorten. The decrease in comet tail abundance caused a proportional increase in actin clouds whereas overexpression of SM22 reversed the actin cloud to comet tail proportions and increased comet tail length, while not influencing EPEC pedestal abundance. Thus, we demonstrate that SM22 plays a role in regulating the transitions and morphological appearance of bacterially generated actin-rich structures during infections. DOI PubMed
80. Chua, MD; Walker, BD; Jin, JP; Guttman, JA. (2018) Calponins Are Recruited to Actin-Rich Structures Generated by Pathogenic Escherichia coli, Listeria, and Salmonella.Anat. Rec. 301 Calponins Are Recruited to Actin-Rich Structures Generated by Pathogenic Escherichia coli, Listeria, and Salmonella
calponin; enteropathogenic Escherichia coli; Listeria monocytogenes; Salmonella enterica serovar Typhimurium; actin
The ingestion of enteropathogenic Escherichia coli (EPEC), Listeria monocytogenes, or Salmonella enterica serovar Typhimurium leads to their colonization of the intestinal lumen, which ultimately causes an array of ailments ranging from diarrhea to bacteremia. Once in the intestines, these microbes generate various actin-rich structures to attach, invade, or move within the host intestinal epithelial cells. Although an assortment of actin-associated proteins has been identified to varying degrees at these structures, the localization of many actin stabilizing proteins have yet to be analyzed. Here, we examined the recruitment of the actin-associated proteins, calponin 1 and 2 at EPEC pedestals, L. monocytogenes actin clouds, comet tails and listeriopods, and S. Typhimurium membrane ruffles. In other systems, calponins are known to bind to and stabilize actin filaments. In EPEC pedestals, calponin 1 was recruited uniformly throughout the structures while calponin 2 was enriched at the apical tip. During L. monocytogenes infections, calponin 1 was found through all the actin-rich structures generated by the bacteria, while calponin 2 was only present within actin-rich structures formed by L. monocytogenes near the host cell membrane. Finally, both calponins were found within S. Typhimuriumgenerated membrane ruffles. Taken together, we have shown that although calponin 1 is recruited to actin-rich structures formed by the three bacteria, calponin 2 is specifically recruited to only membrane-bound actin-rich structures formed by the bacteria. Thus, our findings suggest that calponin 2 is a novel marker for membrane-bound actin structures formed by pathogenic bacteria. (C) 2018 Wiley Periodicals, Inc. DOI PubMed
79. Dhanda, AS; Vogl, AW; Albraiki, SE; Otey, CA; Beck, MR; Guttman, JA. (2018) Palladin Compensates for the Arp2/3 Complex and Supports Actin Structures during Listeria Infections.mBio 9 Palladin Compensates for the Arp2/3 Complex and Supports Actin Structures during Listeria Infections
actin nucleation; actin polymerization; Listeria monocytogenes
Palladin is an important component of motile actin-rich structures and nucleates branched actin filament arrays in vitro. Here we examine the role of palladin during Listeria monocytogenes infections in order to tease out novel functions of palladin. We show that palladin is co-opted by L. monocytogenes during its cellular entry and intracellular motility. Depletion of palladin resulted in shorter and misshapen comet tails, and when actin-or VASP-binding mutants of palladin were overexpressed in cells, comet tails disintegrated or became thinner. Comet tail thinning resulted in parallel actin bundles within the structures. To determine whether palladin could compensate for the Arp2/3 complex, we overexpressed palladin in cells treated with the Arp2/3 inhibitor CK-666. In treated cells, bacterial motility could be initiated and maintained when levels of palladin were increased. To confirm these findings, we utilized a cell line depleted of multiple Arp2/3 complex subunits. Within these cells, L. monocytogenes failed to generate comet tails. When palladin was overexpressed in this Arp2/3 functionally null cell line, the ability of L. monocytogenes to generate comet tails was restored. Using purified protein components, we demonstrate that L. monocytogenes actin clouds and comet tails can be generated ( in a cell-free system) by palladin in the absence of the Arp2/3 complex. Collectively, our results demonstrate that palladin can functionally replace the Arp2/3 complex during bacterial actin-based motility. IMPORTANCE Structures containing branched actin filaments require the Arp2/3 complex. One of the most commonly used systems to study intracellular movement generated by Arp2/3-based actin motility exploits actin-rich comet tails made by Listeria. Using these infections together with live imaging and cell-free protein reconstitution experiments, we show that another protein, palladin, can be used in place of Arp2/3 to form actin-rich structures. Additionally, we show that palladin is needed for the structural integrity of comet tails as its depletion or mutation of critical regions causes dramatic changes to comet tail organization. These findings are the first to identify a protein that can functionally replace the Arp2/3 complex and have implications for all actin-based structures thought to exclusively use that complex. DOI PubMed
78. Dhanda, AS; Warren, KE; Chiu, RH; Guttman, JA. (2018) Cyclophilin A Controls Salmonella Internalization Levels and is Present at E. coli Actin-Rich Pedestals.Anat. Rec. 301 Cyclophilin A Controls Salmonella Internalization Levels and is Present at E. coli Actin-Rich Pedestals
S. Typhimurium; membrane ruffles; EPEC; EHEC; pedestals; cyclophilin A
Salmonella enterica serovar Typhimurium (S. Typhimurium), enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) commandeer the actin cytoskeleton of their host cells as a crucial step in their infectious processes. These pathogens depend on the injection of their own effectors directly into target host cells in order to usurp cellular signaling pathways that lead to morphological actin rearrangements in those cells. Here we show that the PPIase Cyclophilin A (CypA) is a novel component of S. Typhimurium-induced membrane ruffles and functions to restrict bacterial invasion levels, as in cells depleted of CypA, bacterial loads increase. We also demonstrate that CypA requires the EPEC effector Tir as well as pedestal formation for its recruitment to bacterial attachment sites and that its presence at pedestals also holds during EHEC infections. Finally, we demonstrate that CypA is found at lamellipodia; actin-rich structures at the leading edge of motile cells. Our findings further establish CypA as a component of dynamic actin-rich structures formed during bacterial infections and within cells in general. (C) 2018 Wiley Periodicals, Inc. DOI PubMed
77. Vogl, AW; Guttman, JA. (2018) An Introduction to Actin Actin-Rich Structures.Anat. Rec. 301 An Introduction to Actin Actin-Rich Structures
actin; actin-rich structures; microvilli; junction; comet tail
The actin cytoskeleton has long been recognized as a crucial sub-cellular filament system that is responsible for governing fundamental events ranging from cell division and muscle contraction to whole cell motility and the maintenance of tissue integrity. Consequently, it is not surprising that this network is the focus of over 100,000 different manuscripts. Alterations in the actin cytoskeleton lead to an assortment of diseases and serve as a target for a variety of pathogens. Here we have brought together a collection of primary research articles and reviews that underscore the broad influence this filament system has on organisms. (C) 2018 Wiley Periodicals, Inc. DOI PubMed
76. Walker, BD; Chua, MD; Guttman, JA. (2018) Hsc70 is a Component of Bacterially Generated Actin-Rich Structures: An Immunolocalization Study.Anat. Rec. 301 Hsc70 is a Component of Bacterially Generated Actin-Rich Structures: An Immunolocalization Study
Hsc70; Salmonella typhimurium; Listeria monocytogenes; Escherichia coli; actin
Enteropathogenic Escherichia coli (EPEC), Salmonella typhimurium, and Listeria monocytogenes usurp the actin cytoskeleton for their attachment, internalization and transport within and amongst infected cells. To try to gain a greater understanding of the molecular components utilized by these microbes during their infections we previously concentrated actinrich structures generated during EPEC infections (called pedestals) and identified the heat shock cognate 70 protein (Hsc70) as a potential candidate. This multifunctional protein classically acts as a chaperone for the proper folding of a variety of proteins and is involved in uncoating clathrin from coated pits. Here we demonstrated that Hsc70 is recruited to actin structures generated during EPEC, Listeria and Salmonella infections, but not to the same location as clathrin. (C) 2018 Wiley Periodicals, Inc. DOI PubMed
73. Kolappan, S; Lo, KY; Shen, CLJ; Guttman, JA; Craig, L. (2017) Structure of the conserved Francisella virulence protein FvfA.Acta Crystallogr. Sect. D-Struct. Biol. 73: 814-821 Structure of the conserved Francisella virulence protein FvfA
Francisella tularensis; Francisella novicida; membrane proteins; virulence factors; periplasmic proteins; DUF1471
Francisella tularensis is a potent human pathogen that invades and survives in macrophage and epithelial cells. Two identical proteins, FTT_0924 from F. tularensis subsp. tularensis and FTL_1286 from F. tularensis subsp. holarctica LVS, have previously been identified as playing a role in protection of the bacteria from osmotic shock and its survival in macrophages. FTT_0924 has been shown to localize to the inner membrane, with its C-terminus exposed to the periplasm. Here, crystal structures of the F. novicida homologue FTN_0802, which we call FvfA, in two crystal forms are reported at 1.8 angstrom resolution. FvfA differs from FTT_0924 and FTL_1286 by a single amino acid. FvfA has a DUF1471 fold that closely resembles the Escherichia coli outer membrane lipoprotein RscF, a component of a phosphorelay pathway involved in protecting bacteria from outer membrane perturbation. The structural and functional similarities and differences between these proteins and their implications for F. tularensis pathogenesis are discussed. DOI
70. Law, RJ; Law, HT; Scurll, JM; Scholz, R; Santos, AS; Shames, SR; Deng, WY; Croxen, MA; Li, YL; de Hoog, CL; van der Heijden, J; Foster, LJ; Guttman, JA; Finlay, BB. (2016) Quantitative Mass Spectrometry Identifies Novel Host Binding Partners for Pathogenic Escherichia coli Type III Secretion System Effectors.Journal of Proteome Research 15: 1613-1622 Quantitative Mass Spectrometry Identifies Novel Host Binding Partners for Pathogenic Escherichia coli Type III Secretion System Effectors
quantitative mass spectrometry; SILAC; effector proteins; ensconsin; MAP7; enteropathogenic E. coli; enterohemorrhagic E. coli; type III secretion system
Enteropathogenic and enterohemorrhagic Escherichia colt cause enteric diseases resulting in significant morbidity and mortality worldwide. These pathogens remain extracellular and translocate a set of type III secreted effector proteins into host cells to promote bacterial virulence. Effectors manipulate host cell pathways to facilitate infection by interacting with a variety of host targets, yet the binding partners and mechanism of action of many effectors remain elusive. We performed a mass spectrometry screen to identify host targets for a library of effectors. We found five known effector targets and discovered four novel interactions. Interestingly, we identified multiple effectors that interacted with the microtubule associated protein, ensconsin. Using co-immunoprecipitations, we confirmed that NleB1 and EspL interacted with ensconsin in a region that corresponded to its microtubule binding domain. Ensconsin is an essential cofactor of kinesin-1 that is required for intracellular trafficking, and we demonstrated that intracellular trafficking was severely disrupted during wild type EPEC infections but not during infections with Delta nleB1 or Delta espL mutants. Our findings demonstrate the efficacy of quantitative proteomics for identifying effector host protein interactions and suggest that vesicular trafficking is a crucial cellular process that may be targeted by NleB1 and EspL through their interaction with ensconsin. DOI
69. Lo, KY; Visram, S; Vogl, AW; Shen, CLJ; Guttman, JA. (2016) Morphological analysis of Francisella novicida epithelial cell infections in the absence of functional FipA.Cell and Tissue Research 363: 449-459 Morphological analysis of Francisella novicida epithelial cell infections in the absence of functional FipA
Francisella-containing vacuole; FipA; FipB; Vacuole escape; Francisella non-phagocytic infections
Francisella novicida is a surrogate pathogen commonly used to study infections by the potential bioterrorism agent, Francisella tularensis. One of the primary sites of Francisella infections is the liver where > 90 % of infected cells are hepatocytes. It is known that once Francisella enter cells it occupies a membrane-bound compartment, the Francisella-containing vacuole (FCV), from which it rapidly escapes to replicate in the cytosol. Recent work examining the Francisella disulfide bond formation (Dsb) proteins, FipA and FipB, have demonstrated that these proteins are important during the Francisella infection process; however, details as to how the infections are altered in epithelial cells have remained elusive. To identify the stage of the infections where these Dsbs might act during epithelial infections, we exploited a hepatocyte F. novicida infection model that we recently developed. We found that F. novicida Delta fipA-infected hepatocytes contained bacteria clustered within lysosome-associated membrane protein 1-positive FCVs, suggesting that FipA is involved in the escape of F. novicida from its vacuole. Our morphological evidence provides a tangible link as to how Dsb FipA can influence Francisella infections. DOI
65. Law, HT; Chua, M; Moon, KM; Foster, LJ; Guttman, JA. (2015) Mass Spectrometry-Based Proteomics Identification of Enteropathogenic Escherichia coli Pedestal Constituents.Journal of Proteome Research 14: 2520-2527 Mass Spectrometry-Based Proteomics Identification of Enteropathogenic Escherichia coli Pedestal Constituents
Transgelin; cyclophilin A; BDM; epithelial cells; stable isotope labeling
Enteropathogenic Escherichia coli (EPEC) co-opt host signaling pathways and recruit numerous host proteins to motile morphological structures, called pedestals, at sites of bacterial attachment. These pedestals are hallmarks of EPEC-based disease, and the identification and characterization of the functions of pedestal proteins continue to steadily increase. To identify additional constituents in an unbiased manner, we developed a strategy where EPEC pedestals were elongated artificially, severed, and then concentrated prior to their analysis by mass spectrometry (MS)-based proteomics. We identified >90 unique mammalian proteins over multiple experimental trials from our preparations. Seventeen predicted molecules were significantly higher in abundance (p < 0.05) when compared to both the negative controls and sample Means. Validation of two identified proteins (cyclophilin A [nonactin-associated] and transgelin [actin-associated]) by immunolocalization was used to confirm our analysis, and both showed enrichment at EPEC pedestals. The EPEC pedestal concentration technique developed here together with the identification of novel pedestal proteins not only provides a resource for the further characterization of molecular components within these structures but also demonstrates that EPEC pedestals can be used as a model system for the identification of novel functions of proteins not normally thought to be at actin-based structures. DOI
63. Law, HT; Sriram, A; Fevang, C; Nix, EB; Nano, FE; Guttman, JA. (2014) IgIC and PdpA Are Important for Promoting Francisella Invasion and Intracellular Growth in Epithelial Cells.PLOS One 9 IgIC and PdpA Are Important for Promoting Francisella Invasion and Intracellular Growth in Epithelial Cells
The highly infectious bacteria, Francisella tularensis, colonize a variety of organs and replicate within both phagocytic as well as non-phagocytic cells, to cause the disease tularemia. These microbes contain a conserved cluster of important virulence genes referred to as the Francisella Pathogenicity Island (FPI). Two of the most characterized FPI genes, iglC and pdpA, play a central role in bacterial survival and proliferation within phagocytes, but do not influence bacterial internalization. Yet, their involvement in non-phagocytic epithelial cell infections remains unexplored. To examine the functions of IglC and PdpA on bacterial invasion and replication during epithelial cell infections, we infected liver and lung epithelial cells with F. novicida and F. tularensis 'Type B' Live Vaccine Strain (LVS) deletion mutants (Delta iglC and Delta pdpA) as well as their respective gene complements. We found that deletion of either gene significantly reduced their ability to invade and replicate in epithelial cells. Gene complementation of iglC and pdpA partially rescued bacterial invasion and intracellular growth. Additionally, substantial LAMP1-association with both deletion mutants was observed up to 12 h suggesting that the absence of IglC and PdpA caused deficiencies in their ability to dissociate from LAMP1-positive Francisella Containing Vacuoles (FCVs). This work provides the first evidence that IglC and PdpA are important pathogenic factors for invasion and intracellular growth of Francisella in epithelial cells, and further highlights the discrete mechanisms involved in Francisella infections between phagocytic and non-phagocytic cells. DOI PubMed
62. Lo, KYS; Chua, MD; Abdulla, S; Law, HT; Guttman, JA. (2013) Examination of in vitro epithelial cell lines as models for Francisella tularensis non-phagocytic infections.Journal of Microbiological Methods 93: 153-160 Examination of in vitro epithelial cell lines as models for Francisella tularensis non-phagocytic infections
PATHOGENICITY ISLAND; GENOME SEQUENCE; GAP-JUNCTION; SCHU S4; TULAREMIA; MICE; VACCINE
Francisella tularensis (F. tularensis), the causative agent of tularemia, has long been known to invade and occupy non-phagocytic epithelial cells. Many epithelial cell infection models have been developed to study this process; however, due to the lack of consensus on infection methods and precise experimental procedures to evaluate invasion and replication, selection of appropriate models to use based on the literature is challenging. To evaluate in vitro non-phagocytic cell infection models, we chose 8 epithelial cultured cell lines from published models to infect with F. tularensis subspecies novicida (F. novicida) and compared the results to a recently developed model that used the mouse hepatocyte BNL CL2 cell line. We utilized classical gentamicin-based invasion assays to determine total intracellular bacterial loads and employed microscopic examination with staining techniques that distinguished between intracellular and extracellular bacteria to provide an accurate assessment of the proportion of invaded host cells and the degree of bacterial replication. We found that COS-7 cells exhibited the greatest invasion rates; CMT-93 cells contained the largest intracellular bacterial loads; ad HEK-293s were capable of invasion and replication rates at high levels, but required shorter infection incubation times. Although COS-7, CMT-93 and HEK-293 cell lines may be suited to study certain aspects of invasion or replication, we found that BNL CL.2 cells appeared the most appropriate to study the overall pathogenesis of F. novicida when examined in toto. (C) 2013 Elsevier B.V. All rights reserved. DOI
61. Auweter, SD; Yu, HB; Arena, ET; Guttman, JA; Finlay, BB. (2012) Oxysterol-binding protein (OSBP) enhances replication of intracellular Salmonella and binds the Salmonella SPI-2 effector SseL via its N-terminus.Microbes and Infection 14: 148-154 Oxysterol-binding protein (OSBP) enhances replication of intracellular Salmonella and binds the Salmonella SPI-2 effector SseL via its N-terminus
Oxysterol-binding protein; OSBP; Salmonella enterica serovar Typhimurium; Type three secretion; SPI-2; SseL
Effectors translocated into the host cell by Salmonella enterica serovar Typhimurium are critical for bacterial virulence. For many effectors, the mechanisms of their interactions with host pathways are not yet understood. We have recently found an interaction between the SPI-2 effector SseL and oxysterol-binding protein (OSBP). We show here that SseL binds the N-terminus of OSBP and that S. Typhimurium infection results in redistribution of OSBP. We furthermore demonstrate that OSBP is required for efficient replication of intracellular S. Typhimurium. This suggests that S. Typhimurium hijacks OSBP-dependent pathways to benefit its intracellular life-style, possibly by SseL- and OSBP-mediated manipulation of host lipid metabolism. (C) 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. DOI
60. Law, HT; Bonazzi, M; Jackson, J; Cossart, P; Guttman, JA. (2012) Nexilin is a dynamic component of Listeria monocytogenes and enteropathogenic Escherichia coli actin-rich structures.Cellular Microbiology 14: 1097-1108 Nexilin is a dynamic component of Listeria monocytogenes and enteropathogenic Escherichia coli actin-rich structures
The bacterial pathogens Listeria monocytogenes and enteropathogenic Escherichia coli (EPEC) generate motile actin-rich structures (comet tails and pedestals) as part of their infectious processes. Nexilin, an actin-associated protein and a component of focal adhesions, has been suggested to be involved in actin-based motility. To determine whether nexilin is commandeered during L. monocytogenes and EPEC infections, we infected cultured cells and found that nexilin is crucial for L. monocytogenes invasion as levels of internalized bacteria were significantly decreased in nexilin-targeted siRNA-treated cells. In addition, nexilin is a component of the machinery that drives the formation of L. monocytogenes comet tails and EPEC pedestals. Nexilin colocalizes with stationary bacteria and accumulates at the distal portion of comet tails and pedestals of motile bacteria. We also show that nexilin is crucial for efficient comet tail formation as cells pre-treated with nexilin siRNA generate malformed comet tails, whereas nexilin is dispensable during EPEC pedestal generation. These findings demonstrate that nexilin is required for efficient infection with invasive and adherent bacteria and is key to the actin-rich structures these microbes generate. DOI
59. Lin, AE; Guttman, JA. (2012) Lack of Tir Ubiquitylation Contributes to Enteropathogenic E. coli Remaining Extracellular During Nonphagocytic Cell Infections.Anatomical Record-Advances in Integrative Anatomy and Evolutionary Biology 295: 1230-1238 Lack of Tir Ubiquitylation Contributes to Enteropathogenic E. coli Remaining Extracellular During Nonphagocytic Cell Infections
enteropathogenic E; coli; ubiquitylation; translocated intimin receptor; pedestal; internalization
Enteropathogenic Escherichia coli (EPEC) is an extracellular pathogen that alters many host subcellular components during its infectious processes. We have previously shown that EPEC hijacks a large assortment of host cell endocytic components and uses these proteins to form protruding structures called pedestals rather than triggering internalization of the bacteria. Other invasive pathogens that also recruit similar endocytic components have been shown to enter their host cells on the ubiquitylation of their host cell receptors. Therefore, we hypothesize that EPEC remains extracellular by maintaining its receptor, translocated intimin receptor (Tir), in an unubiquitylated state. Using immunoprecipitation-Western blots, we demonstrate no association of ubiquitin with Tir. To further elucidate the effect Tir ubiquitylation would have on EPEC during their infections, we engineered Tir-ubiquitin fusion constructs, expressed them in host epithelial cells, and infected them with ?tir EPEC. We found these cells induced a significant increase in EPEC invasion as compared with cells that expressed the Tir construct that lacked ubiquitin conjugation. Our results indicate that the lack of EPEC receptor ubiquitylation is a contributing factor that these microbes use to prevent their internalization into epithelial cells. Anat Rec, 2012. (C) 2012 Wiley Periodicals, Inc. DOI
58. Lin, AE; Guttman, JA. (2012) The Escherichia coli adherence factor plasmid of enteropathogenic Escherichia coli causes a global decrease in ubiquitylated host cell proteins by decreasing ubiquitin E1 enzyme expression through host aspartyl proteases.International Journal of Biochemistry & Cell Biology 44: 2223-2232 The Escherichia coli adherence factor plasmid of enteropathogenic Escherichia coli causes a global decrease in ubiquitylated host cell proteins by decreasing ubiquitin E1 enzyme expression through host aspartyl proteases
Ubiquitin; EPEC; JPN15; EAF plasmid
Ubiquitylation is a widespread post-translational global regulatory system that is essential for the proper functioning of various cellular events. Recent studies have shown that certain types of Escherichia coil can exploit specific aspects of the ubiquitylation system to influence downstream targets. Despite these findings, examination of the effects pathogenic E. coli have on the overall host ubiquitylation system remain unexplored. To study the impact that pathogenic E. coli have on the ubiquitylation levels of host proteins during infections, we analyzed the entire ubiquitylation system during enteropathogenic E. coil infections of cultured cells. We found that these microbes caused a dramatic decrease in ubiquitylated host proteins during these infections. This occurred with a concomitant reduction in the expression of essential El activating enzymes in the host, which are integral for the initiation of the ubiquitylation cascade. Control of host El enzyme levels was dependent on the E. coli adherence factor plasmid which acted on host aspartyl proteases within enteropathogenic E. coil. Hijacking of the ubiquitylation system did not require the plasmid-encoded regulator or bundle forming pilus expression, as enteropathogenic E. coil mutated in those factors did not revert the ubiquitylation of host proteins or the abundance of El enzyme proteins to uninfected levels. Our work shows that E. coil have developed strategies to usurp post-translational systems by targeting crucial enzymes. The ability of enteropathogenic E. coil to inactivate host protein ubiquitylation could enable more efficient effector protein functionality, providing increased bacterial control of host cells during enteropathogenic E. coil pathogenesis. (C) 2012 Elsevier Ltd. All rights reserved. DOI
57. Ruetz, TJ; Lin, AE; Guttman, JA. (2012) Shigella flexneri utilize the spectrin cytoskeleton during invasion and comet tail generation.BMC Microbiology 12 Shigella flexneri utilize the spectrin cytoskeleton during invasion and comet tail generation
Background: The spectrin cytoskeleton is emerging as an important host cell target of enteric bacterial pathogens. Recent studies have identified a crucial role for spectrin and its associated proteins during key pathogenic processes of Listeria monocytogenes and Salmonella Typhimurium infections. Here we investigate the involvement of spectrin cytoskeletal components during the pathogenesis of the invasive pathogen Shigella flexneri. Results: Immunofluorescent microscopy reveals that protein 4.1 (p4.1), but not adducin or spectrin, is robustly recruited to sites of S. flexneri membrane ruffling during epithelial cell invasion. Through siRNA-mediated knockdowns, we identify an important role for spectrin and the associated proteins adducin and p4.1 during S. flexneri invasion. Following internalization, all three proteins are recruited to the internalized bacteria, however upon generation of actin-rich comet tails, we observed spectrin recruitment to those structures in the absence of adducin or p4.1. Conclusion: These findings highlight the importance of the spectrin cytoskeletal network during S. flexneri pathogenesis and further demonstrate that pathogenic events that were once thought to exclusively recruit the actin cytoskeletal system require additional cytoskeletal networks. DOI
56. Ruetz, TJ; Lin, AEJ; Guttman, JA. (2012) Enterohaemorrhagic Escherichia coli requires the spectrin cytoskeleton for efficient attachment and pedestal formation on host cells.Microbial Pathogenesis 52: 149-156 Enterohaemorrhagic Escherichia coli requires the spectrin cytoskeleton for efficient attachment and pedestal formation on host cells
EHEC; EPEC; Spectrin cytoskeleton
Recent work has demonstrated that the spectrin cytoskeleton is a host cell target, exploited during intestinal bacterial disease. Here we show that the highly virulent intestinal pathogen enterohaemorrhagic Escherichia coil (EHEC) is also reliant upon the spectrin cytoskeleton during key pathogenic events. Immunofluorescent microscopy demonstrated that the core components of the spectrin cytoskeleton (spectrin, adducin, and protein 4.1 [p4.1]) are recruited to sites of EHEC attachment and localized at pedestal structures along with the EHEC pedestal specific proteins IRSp53 and IRTKS. Further studies involving siRNA-mediated knockdowns of spectrin, adducin, or p4.1 revealed that those proteins are needed for efficient docking of EHEC to host cells, are involved in recruiting IRSp53 to the pedestal and are necessary for pedestal formation. These findings identify the spectrin cytoskeleton as a major host cell cytoskeletal network involved in critical EHEC pathogenic events. (C) 2011 Elsevier Ltd. All rights reserved. DOI
55. Ruetz, TJ; Vogl, AW; Guttman, JA. (2012) Detailed Examination of Cytoskeletal Networks Within Enteropathogenic Escherichia coli Pedestals.Anatomical Record-Advances in Integrative Anatomy and Evolutionary Biology 295: 201-207 Detailed Examination of Cytoskeletal Networks Within Enteropathogenic Escherichia coli Pedestals
spectrin; actin; enteropathogenic Escherichia coli
Enteropathogenic Escherichia coli (EPEC) manipulate the cytoskeleton of host intestinal epithelial cells, producing membrane protrusions termed pedestals that the bacteria reside on throughout the course of their infections. By definition pedestals are actin-based structures, however recent work has identified the spectrin cytoskeleton as a necessary component of EPEC pedestals. Here, we investigated the detailed arrangement of the spectrin and actin cytoskeletons within these structures. Immunofluorescent imaging revealed that the spectrin network forms a peripheral cage around actin at the membranous regions of pedestals. Myosin S1 fragment decorated actin filaments examined by electron microscopy demonstrated that actin filaments orientate with their fast-growing barbed ends toward the lateral membranes of EPEC pedestals. These findings provide a detailed descriptive analysis, which further illustrate the spectrin cytoskeletal organization within these structures. Anat Rec, 2012. (C) 2011 Wiley Periodicals, Inc. DOI
53. Arena, ET; Auweter, SD; Antunes, LCM; Vogl, AW; Han, J; Guttman, JA; Croxen, MA; Menendez, A; Covey, SD; Borchers, CH; Finlay, BB. (2011) The Deubiquitinase Activity of the Salmonella Pathogenicity Island 2 Effector, SseL, Prevents Accumulation of Cellular Lipid Droplets.Infection and Immunity 79: 4392-4400 The Deubiquitinase Activity of the Salmonella Pathogenicity Island 2 Effector, SseL, Prevents Accumulation of Cellular Lipid Droplets
To cause disease, Salmonella enterica serovar Typhimurium requires two type III secretion systems that are encoded by Salmonella pathogenicity islands 1 and 2 (SPI-1 and -2). These secretion systems serve to deliver specialized proteins (effectors) into the host cell cytosol. While the importance of these effectors to promote colonization and replication within the host has been established, the specific roles of individual secreted effectors in the disease process are not well understood. In this study, we used an in vivo gallbladder epithelial cell infection model to study the function of the SPI-2-encoded type III effector, SseL. The deletion of the sseL gene resulted in bacterial filamentation and elongation and the unusual localization of Salmonella within infected epithelial cells. Infection with the Delta sseL strain also caused dramatic changes in host cell lipid metabolism and led to the massive accumulation of lipid droplets in infected cells. This phenotype was directly attributable to the deubiquitinase activity of SseL, as a Salmonella strain carrying a single point mutation in the catalytic cysteine also resulted in extensive lipid droplet accumulation. The excessive buildup of lipids due to the absence of a functional sseL gene also was observed in murine livers during S. Typhimurium infection. These results suggest that SseL alters host lipid metabolism in infected epithelial cells by modifying the ubiquitination patterns of cellular targets. DOI
52. Law, HT; Lin, AEJ; Kim, Y; Quach, B; Nano, FE; Guttman, JA. (2011) Francisella tularensis Uses Cholesterol and Clathrin-Based Endocytic Mechanisms to Invade Hepatocytes.Scientific Reports 1 Francisella tularensis Uses Cholesterol and Clathrin-Based Endocytic Mechanisms to Invade Hepatocytes
Francisella tularensis are highly infectious microbes that cause the disease tularemia. Although much of the bacterial burden is carried in non-phagocytic cells, the strategies these pathogens use to invade these cells remains elusive. To examine these mechanisms we developed two in vitro Francisella-based infection models that recapitulate the non-phagocytic cell infections seen in livers of infected mice. Using these models we found that Francisella novicida exploit clathrin and cholesterol dependent mechanisms to gain entry into hepatocytes. We also found that the clathrin accessory proteins AP-2 and Eps15 co-localized with invading Francisella novicida as well as the Francisella Live Vaccine Strain (LVS) during hepatocyte infections. Interestingly, caveolin, a protein involved in the invasion of Francisella in phagocytic cells, was not required for non-phagocytic cell infections. These results demonstrate a novel endocytic mechanism adopted by Francisella and highlight the divergence in strategies these pathogens utilize between non-phagocytic and phagocytic cell invasion. DOI
51. Lin, AE; Benmerah, A; Guttman, JA. (2011) Eps15 and Epsin1 Are Crucial for Enteropathogenic Escherichia coli Pedestal Formation Despite the Absence of Adaptor Protein 2.Journal of Infectious Diseases 204: 695-703 Eps15 and Epsin1 Are Crucial for Enteropathogenic Escherichia coli Pedestal Formation Despite the Absence of Adaptor Protein 2
Enteropathogenic Escherichia coli (EPEC) are primarily extracellular pathogens that generate actin-rich structures known as pedestals during their pathogenesis. Surprising evidence has demonstrated that despite maintaining an extracellular location, EPEC require the endocytic protein, clathrin, for pedestal formation. To evaluate the strategies EPEC use to usurp endocytic machinery, we investigated the roles of a number of clathrin-coated pits components, adaptor protein 2 (AP-2), Eps15 and epsin1, during EPEC infections. We demonstrated that in conjunction with clathrin, pedestal formation also required the recruitment of Eps15 and epsin1 but not AP-2. Because AP-2 orchestrates the recruitment of clathrin, Eps15, and epsin1, as well as other adaptors, during assembly of clathrin-coated pits at the plasma membrane, our findings reveal a novel internalization subversion strategy employed by EPEC. These results further emphasize the recent paradigm that endocytic proteins are important for EPEC-mediated disease. DOI
50. Ruetz, T; Cornick, S; Guttman, JA. (2011) The Spectrin Cytoskeleton Is Crucial for Adherent and Invasive Bacterial Pathogenesis.PLOS One 6(5):e19940 The Spectrin Cytoskeleton Is Crucial for Adherent and Invasive Bacterial Pathogenesis
Various enteric bacterial pathogens target the host cell cytoskeletal machinery as a crucial event in their pathogenesis. Despite thorough studies detailing strategies microbes use to exploit these components of the host cell, the role of the spectrin-based cytoskeleton has been largely overlooked. Here we show that the spectrin cytoskeleton is a host system that is hijacked by adherent (Entropathogenic Escherichia coli [EPEC]), invasive triggering (Salmonella enterica serovar Typhimurium [S. Typhimurium]) and invasive zippering (Listeria monocytogenes) bacteria. We demonstrate that spectrin cytoskeletal proteins are recruited to EPEC pedestals, S. Typhimurium membrane ruffles and Salmonella containing vacuoles (SCVs), as well as sites of invasion and comet tail initiation by L. monocytogenes. Spectrin was often seen co-localizing with actin filaments at the cell periphery, however a disconnect between the actin and spectrin cytoskeletons was also observed. During infections with S. Typhimurium Delta sipA, actin-rich membrane ruffles at characteristic sites of bacterial invasion often occurred in the absence of spectrin cytoskeletal proteins. Additionally, early in the formation of L. monocytogenes comet tails, spectrin cytoskeletal elements were recruited to the surface of the internalized bacteria independent of actin filaments. Further studies revealed the presence of the spectrin cytoskeleton during SCV and Listeria comet tail formation, highlighting novel cytoplasmic roles for the spectrin cytoskeleton. SiRNA targeted against spectrin and the spectrin-associated proteins severely diminished EPEC pedestal formation as well as S. Typhimurium and L. monocytogenes invasion. Ultimately, these findings identify the spectrin cytoskeleton as a ubiquitous target of enteric bacterial pathogens and indicate that this cytoskeletal system is critical for these infections to progress. DOI
49.Guttman, JA; Lin, AE; Veiga, E; Cossart, P; Finlay, BB. (2010) Role for CD2AP and Other Endocytosis-Associated Proteins in Enteropathogenic Escherichia coli Pedestal Formation.Infection and Immunity 78: 3316-3322 Role for CD2AP and Other Endocytosis-Associated Proteins in Enteropathogenic Escherichia coli Pedestal Formation
Enteropathogenic Escherichia coli (EPEC) strains are extracellular pathogens that generate actin-rich structures (pedestals) beneath the adherent bacteria as part of their virulence strategy. Pedestals are hallmarks of EPEC infections, and their efficient formation in vitro routinely requires phosphorylation of the EPEC effector protein Tir at tyrosine 474 (Y474). This phosphorylation results in the recruitment and direct attachment of the host adaptor protein Nck to Tir at Y474, which is utilized for actin nucleation through a downstream N-WASP-Arp2/3-based mechanism. Recently, the endocytic protein clathrin was demonstrated to be involved in EPEC pedestal formation. Here we examine the organization of clathrin in pedestals and report that CD2AP, an endocytosis-associated and cortactin-binding protein, is a novel and important component of EPEC pedestal formation that also utilizes Y474 phosphorylation of EPEC Tir. We also demonstrate the successive recruitment of Nck and then clathrin prior to actin polymerization at pedestals during the Nck-dependent pathway of pedestal formation. This study further demonstrates that endocytic proteins are key components of EPEC pedestals and suggests a novel endocytosis subversion strategy employed by these extracellular bacteria. DOI
48.Guttman, JA; Lin, AEJ; Li, YL; Bechberger, J; Naus, CC; Vogl, AW; Finlay, BB. (2010) Gap junction hemichannels contribute to the generation of diarrhoea during infectious enteric disease.Gut 59: 218-226 Gap junction hemichannels contribute to the generation of diarrhoea during infectious enteric disease
Objective The attaching and effacing (A/E) pathogens enterohaemorrhagic Escherichia coli, enteropathogenic E coli and Citrobacter rodentium colonise intestinal tracts, attach to enterocytes, collapse infected cell microvilli and alter numerous host cell processes during infection. Enterocyte alterations result in numerous small molecules being released from host cells that likely contribute to diarrhoeal phenotypes observed during these infections. One possible route for small molecules to be released from intestinal cells may be through functional gap junction hemichannels. Here we examine the involvement of these hemichannels during the diarrhoeal disease caused by A/E pathogens in vivo. Design Mice were infected with the diarrhoea-causing murine A/E pathogen C rodentium for 7 days. Connexin43 (Cx43) protein levels and immunolocalisation in the colon were initially used to determine alterations during A/E bacterial infections in vivo. Connexin mimetic peptides and connexin permeable tracer molecules were used to gage the presence and function of unpaired connexin hemichannels. The role of Cx43 in diarrhoea generation was assessed by comparing infections of wild-type mice to Cx43 mutant mice and determining the water abundance in the colonic luminal material. Results We demonstrate that Cx43 protein levels are increased in colonocytes during in vivo A/E bacterial infections, resulting in functionally open connexon hemichannels in apical membranes of infected cells. Moreover, infected Cx43 +/- mice do not suffer from diarrhoeal disease. Conclusions This study provides the first evidence that functional connexon hemichannels can occur in the intestine and are a novel molecular mechanism of water release during infectious diarrhoea. DOI
47. Lin, AEJ; Guttman, JA. (2010) Hijacking the endocytic machinery by microbial pathogens.Protoplasma 244: 75-90 Hijacking the endocytic machinery by microbial pathogens
Clathrin; Caveolin; Cholesterol; Toxin; Virus; Bacteria
Understanding the mechanisms that microbes exploit to invade host cells and cause disease is crucial if we are to eliminate their threat. Although pathogens use a variety of microbial factors to trigger entry into non-phagocytic cells, their targeting of the host cell process of endocytosis has emerged as a common theme. To accomplish this, microbes often rewire the normal course of particle internalization, frequently usurping theoretical maximal sizes to permit entry and reconfiguring molecular components that were once thought to be required for vesicle formation. Here, we discuss recent advances in our understanding of how toxins, viruses, bacteria, and fungi manipulate the host cell endocytic machinery to generate diseases. Additionally, we will reveal the advantages of using these organisms to expand our general knowledge of endocytic mechanisms in eukaryotic cells. DOI
46. Shames, SR; Deng, WY; Guttman, JA; de Hoog, CL; Li, YL; Hardwidge, PR; Sham, HP; Vallance, BA; Foster, LJ; Finlay, BB. (2010) The pathogenic E-coli type III effector EspZ interacts with host CD98 and facilitates host cell prosurvival signalling.Cellular Microbiology 12: 1322-1339 The pathogenic E-coli type III effector EspZ interacts with host CD98 and facilitates host cell prosurvival signalling
Cell Biology; Microbiology
Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC respectively) are diarrhoeal pathogens that cause the formation of attaching and effacing (A/E) lesions on infected host cells. These pathogens encode a type III secretion system (T3SS) used to inject effector proteins directly into host cells, an essential requirement for virulence. In this study, we identified a function for the type III secreted effector EspZ. Infection with EPEC Delta espZ caused increased cytotoxicity in HeLa and MDCK cells compared with wild-type EPEC, and expressing espZ in cells abrogated this effect. Using yeast two-hybrid, proteomics, immunofluorescence and co-immunoprecipitation, it was demonstrated that EspZ interacts with the host protein CD98, which contributes to protection against EPEC-mediated cytotoxicity. EspZ enhanced phosphorylation of focal adhesion kinase (FAK) and AKT during infection with EPEC, but CD98 only appeared to facilitate FAK phosphorylation. This study provides evidence that EspZ and CD98 promote host cell survival mechanisms involving FAK during A/E pathogen infection. DOI
45.Guttman, JA; Finlay, BB. (2009) Tight junctions as targets of infectious agents.Biochimica et Biophysica Acta-Biomembranes 1788: 832-841 Tight junctions as targets of infectious agents
ENTEROPATHOGENIC ESCHERICHIA-COLI; ZONULA OCCLUDENS TOXIN; CLOSTRIDIUM-PERFRINGENS ENTEROTOXIN; INTESTINAL EPITHELIAL-CELLS; HEPATITIS-C VIRUS; HELICOBACTER-PYLORI CAGA; BARRIER FUNCTION; PARACELLULAR PERMEABILITY; ADHESION MOLECULE; COXSACKIEVIRUS ENTRY
The epithelial barrier is a critical border that segregates luminal material from entering tissues. Essential components of this epithelial fence are physical intercellular structures termed tight junctions. These junctions use a variety of transmembrane proteins coupled with cytoplasmic adaptors, and the actin cytoskeleton, to attach adjacent cells together thereby forming intercellular seals. Breaching of this barrier has profound effects on human health and disease, as barrier deficiencies have been linked with the onset of inflammation, diarrhea generation and pathogenic effects. Although tight junctions efficiently restrict most microbes from penetrating into deeper tissues and contain the microbiota, some pathogens have developed specific strategies to alter or disrupt these structures as part of their pathogenesis, resulting in either pathogen penetration, or other consequences such as diarrhea. Understanding the strategies that microorganisms use to commandeer the functions of tight junctions is an active area of research in microbial pathogenesis. In this review we highlight and overview the tactics bacteria and viruses use to alter tight junctions during disease. Additionally, these studies have identified novel tight junction protein functions by using pathogens and their virulence factors as tools to study the cell biology of junctional structures. (C) 2008 Elsevier B.V. All rights reserved DOI
44. Menendez, A; Arena, ET; Guttman, JA; Thorson, L; Vallance, BA; Vogl, W; Finlay, BB. (2009) Salmonella Infection of Gallbladder Epithelial Cells Drives Local Inflammation and Injury in a Model of Acute Typhoid Fever.Journal of Infectious Diseases 200: 1703-1713 Salmonella Infection of Gallbladder Epithelial Cells Drives Local Inflammation and Injury in a Model of Acute Typhoid Fever
ACUTE ACALCULOUS CHOLECYSTITIS; IN-VIVO; PEYERS-PATCHES; BILIARY-TRACT; TYPHIMURIUM; BILE; MACROPHAGES; RESISTANCE; IDENTIFICATION; PERFORATION
The gallbladder is often colonized by Salmonella during typhoid fever, yet little is known about bacterial pathogenesis in this organ. With use of a mouse model of acute typhoid fever, we demonstrate that Salmonella infect gallbladder epithelial cells in vivo. Bacteria in the gallbladder showed a unique behavior as they replicated within gallbladder epithelial cells and remained confined to those cells without translocating to the mucosa. Infected gallbladders showed histopathological damage characterized by destruction of the epithelium and massive infiltration of neutrophils, accompanied by a local increase of proinflammatory cytokines. Damage was determined by the ability of Salmonella to invade gallbladder epithelial cells and was independent of high numbers of replication-competent, although invasion-deficient, bacteria in the lumen. Our results establish gallbladder epithelial cells as a novel niche for in vivo replication of Salmonella and reveal the involvement of these cells in the pathogenesis of Salmonella in the gallbladder during the course of acute typhoid fever. DOI
43. Valdez, Y; Grassl, GA; Guttman, JA; Coburn, B; Gros, P; Vallance, BA; Finlay, BB. (2009) Nramp1 drives an accelerated inflammatory response during Salmonella-induced colitis in mice.Cellular Microbiology 11: 351-362 Nramp1 drives an accelerated inflammatory response during Salmonella-induced colitis in mice
ENTERICA SEROTYPE TYPHIMURIUM; SLC11A1 FORMERLY NRAMP1; NATURAL-RESISTANCE; SEROVAR TYPHIMURIUM; INTRACELLULAR PARASITES; DENDRITIC CELLS; BCG/ITY/LSH LOCUS; CANDIDA-ALBICANS; GENETIC-CONTROL; C57BL/6J MICE
A recently developed model for enterocolitis in mice involves pre-treatment with the antibiotic streptomycin prior to infection with Salmonella enterica serovar Typhimurium (S. Typhimurium). The contribution of Nramp1/Slc11a1 protein, a critical host defence mechanism against S. Typhimurium, to the development of inflammation in this model has not been studied. Here, we analysed the impact of Nramp1 expression on the early development of colitis using isogenic Nramp1(+/+) and Nramp1(-/-) mice. We hypothesized that Nramp1 acts by rapidly inducing an inflammatory response in the gut mucosa creating an antibacterial environment and limiting spread of S. Typhimurium to systemic sites. We observed that Nramp1(+/+) mice showed lower numbers of S. Typhimurium in the caecum compared with Nramp1(-/-) mice at all times analysed. Acute inflammation was much more pronounced in Nramp1(+/+) mice 1 day after infection. The effect of Nramp1 on development of colitis was characterized by higher secretion of the pro-inflammatory cytokines IFN-gamma, TNF-alpha and MIP-1 alpha and a massive infiltration of neutrophils and macrophages, compared with Nramp1(-/-) animals. These data show that an early and rapid inflammatory response results in protection against pathological effects of S. Typhimurium infection in Nramp1(+/+) mice. DOI
42. Young, JS; Guttman, JA; Vaid, KS; Shahinian, H; Vogl, AW. (2009) Cortactin (CTTN), N-WASP (WASL), and Clathrin (CLTC) Are Present at Podosome-Like Tubulobulbar Complexes in the Rat Testis.Biology of Reproduction 80: 153-161 Cortactin (CTTN), N-WASP (WASL), and Clathrin (CLTC) Are Present at Podosome-Like Tubulobulbar Complexes in the Rat Testis
cortactin (CTTN); clathrin (CLTC); male reproductive tract; N-WASP (WASL); Sertoli cells; spermatogenesis; testis; tubulobulbar complexes
Tubulobulbar complexes are actin filament-rich plasma membrane protrusions that form at intercellular junctions in the seminiferous epithelium of the mammalian testis. They are proposed to internalize intact junctions during sperm release and during the translocation of spermatocytes through basal junction complexes between neighboring Sertoli cells. Tubulobulbar complexes morphologically resemble podosomes found at cell/substrate attachments in other systems. In this study we probe apical tubulobulbar complexes in fixed epithelial fragments and fixed frozen sections of rat testis for two key actin-related components found at podosomes, and for the endocytosis-related protein clathrin. N-WASP and cortactin, two regulators of actin network assembly known to be components of podosomes, are concentrated at tubulobulbar complexes. Clathrin-positive structures occur in Sertoli cell regions containing tubulobulbar complexes when analyzed by immunofluorescence microscopy and occur at the ends of the complexes when evaluated by immunoelectron microscopy. Our results are consistent with the conclusion that tubulobulbar complexes are podosome-like structures. We propose that the formation of tubulobulbar complexes may be clathrin initiated and that their growth is due to the dendritic assembly of a membrane-related actin network. DOI
41. Young, JS; Guttman, JA; Vaid, KS; Vogl, AW. (2009) Tubulobulbar Complexes Are Intercellular Podosome-Like Structures That Internalize Intact Intercellular Junctions During Epithelial Remodeling Events in the Rat Testis.Biology of Reproduction 80: 162-174 Tubulobulbar Complexes Are Intercellular Podosome-Like Structures That Internalize Intact Intercellular Junctions During Epithelial Remodeling Events in the Rat Testis
intercellular junctions; podosomes; Sertoli cells; spermatogenesis; testis; tubulobulbar complexes
Tubulobulbar complexes are actin-related double-membrane projections that resemble podosomes in other systems and form at intercellular junctions in the seminiferous epithelium of the mammalian testis. They are proposed to internalize intact junctions during sperm release and during the translocation of spermatocytes through basal junction complexes between neighboring Sertoli cells. In this study we probe apical tubulobulbar complexes in fixed epithelial fragments and fixed frozen sections of rat and mouse testes for junction molecules reported to be present at apical sites of attachment (ectoplasmic specializations) between Sertoli cells and spermatids. The adhesion molecules nectin 2 (PVRL2), nectin 3 (PVRL3) and alpha 6 integrin (ITGA6) are present in the elongate parts of tubulobulbar complexes and concentrated at their distal ends. Tubulobulbar complexes contain cortactin (CTTN), a key component of podosomes, and vesicles at the distal ends of tubulobulbar complexes that contain junction molecules are related to early endosome antigen (EEA1). N-cadherin (CDH2), a protein reported to be present at ectoplasmic specializations, is not localized to these unique junctions or to tubulobulbar complexes but, rather, is primarily concentrated at desmosomes in basal regions of the epithelium. Our results are consistent with the conclusion that tubulobulbar complexes are podosome-like structures that are responsible for internalizing intact intercellular junctions during spermatogenesis. DOI
39. Champion OL, Valdez Y, Thorson L, Guttman JA, Menendez A, Gaynor EC, Finlay BB. (2008) A murine intraperitoneal infection model reveals that host resistance to Campylobacter jejuni is Nramp1 dependent.Microbes and Infection 10(8):922-7. Epub 2008 May 14. A murine intraperitoneal infection model reveals that host resistance to Campylobacter jejuni is Nramp1 dependent.
We tested the hypothesis that host resistance to Campylobacter jejuni is Nramp I dependent. Following intraperitoneal (IP) inoculation of Nramp1+/+ and isogenic Nramp1-deficient (Nramp1-/-) mice C. jejuni primarily associated with mac1-positive cells in liver tissue. A significant reduction of C. jejuni was observed in Nramp1 +/+ mice 4 days post-infection (PI) (liver) and 8 days PI cecum-colon. In contrast, Nramp1 -/- mice showed no significant reduction of C. jejuni and instead had a chronic inflammatory response and significant histopathological lesions 30 days PI. Differential cytokine profiles were observed in C. jejuni infected Nramp1 +/+ and Nramp1 -/- primary dendritic cells. Taken together these data indicate that Nramp I is critical for host resistance to C. jejuni. (c) 2008 Elsevier Masson SAS. All rights reserved. DOI
36. Valdez, Y; Diehl, GE; Vallance, BA; Grassl, GA; Guttman, JA; Brown, NF; Rosenberger, CM; Littman, DR; Gros, P; Finlay, BB. (2008) Nramp1 expression by dendritic cells modulates inflammatory responses during Salmonella Typhimurium infection.Cellular Microbiology 10: 1646-1661 Nramp1 expression by dendritic cells modulates inflammatory responses during Salmonella Typhimurium infection
Host resistance against Salmonella enterica serovar Typhimurium (S. Typhimurium) is mediated by natural resistance-associated macrophage protein 1 (Nramp1/Slc11a1). Nramp1 is critical to host defence, as mice lacking Nramp1 fail to control bacterial replication and succumb to low doses of S. Typhimurium. Despite this crucial role, the mechanisms underlying Nramp1's protective effects are unclear. Dendritic cells (DCs) that sample the intestinal lumen are among the first cells encountered by S. Typhimurium following oral infection and act as a conduit for S. Typhimurium to cross the intestinal epithelial barrier. We report that DCs, including intestinal, splenic and bone marrow-derived DCs (BMDCs), express Nramp1 protein. In the small intestine, Nramp1 expression is greater in a subset of DCs (CD11c(+)CD103(-)) characterized by the elevated expression of pro-inflammatory cytokines in response to bacterial products. While Nramp1 expression did not affect S. Typhimurium replication in BMDCs, infected Nramp1+/+ BMDCs and intestinal CD11c(+)CD103(-) DCs secreted more inflammatory cytokines (IL-6, IL-12 and TNF-alpha) than Nramp1-/-, suggesting that Nramp1 expression may promote a more rapid inflammatory response following infection. Collectively, these findings reveal a new role for DCs and Nramp1 in modulating the host inflammatory response to S. Typhimurium. DOI
35. Vogl, AW; Vaid, KS; Guttman, JA. (2008) The Sertoli Cell Cytoskeleton.Molecular Mechanisms in Spermatogenesis 636: 186-211 The Sertoli Cell Cytoskeleton
The cytoskeleton of terminally differentiated mammalian Sertoli cells is one of the most elaborate of those that have been described for cells in tissues. Actin filaments, intermediate Filaments and microtubules have distinct patterns of distribution that change during the cyclic process of spermatogenesis. Each of the three major cytoskeletal elements is either concentrated at or related in part to intercellular junctions. Actin filaments arc concentrated in unique structures termed ectoplasmic specializations that function in intercellular adhesion, and at tubulobulbar complexes that are thought to be involved with junction internalization during sperm release and movement of spermatocytes; through basal junctions between neighboring Sertoi cells. Intermediate filaments occur in a perinuclear network which has peripheral extensions to desmosome-like junctions with adjacent cells and to small hemidesmosome-likc attachments to the basal lamina. Unlike in most other epithelia where the intermediate filaments are of the keratin type, intermediate filaments in mature Sertoli cells are of the vimentin type. The function of intermediate filaments in Sertoli cells is not entirely clear; however, the pattern of filament distribution and the limited experimental data available arc consistent with a role in maintaining tissue integrity when the epithelium is mechanically stressed. Microtubules are abundant in Sertoli cells and are predominantly oriented parallel to the long axis of the cell. Microtubules are involved with maintaining the columnar shape of Sertoli cells, with transporting and positioning organclles in the cytoplasm, and with secreting seminiferous tubule fluid. In addition, microtubule-based transport machinery is coupled to intercellular junctions to translocate and position adjacent spermatids in the epithelium. Although the cytoskeleton of Sertoli cells has structural and functional properties common to cells generally, there are a number of properties that arc unique and that appear related to processes fundamental to spermatogenesis and to interfacing somatic cells both with similar neighboring somatic cells and with differentiating cells of the germ cell line.
34. Bhavsar A, Guttman, JA., Finlay, BB. (2007) Manipulation of host-cell pathways by bacterial pathogens.Nature 449:827-834. Manipulation of host-cell pathways by bacterial pathogens.
Bacterial pathogens operate by attacking crucial intracellular pathways in their hosts. These pathogens
usually target more than one intracellular pathway and often interact at several points in each of these
pathways to commandeer them fully. Although different bacterial pathogens tend to exploit similar pathway
components in the host, the way in which they ‘hijack’ host cells usually differs. Knowledge of how pathogens
target distinct cytoskeletal components and immune-cell signalling pathways is rapidly advancing, together
with the understanding of bacterial virulence at a molecular level. Studying how these bacterial pathogens
subvert host-cell pathways is central to understanding infectious disease. PDF DOI
33.Guttman JA, Vaid KS, Vogl AW. (2007) Enrichment and disassembly of ectoplasmic specializations in the rat testis.Methods in Molecular Biology 392:159-70. Enrichment and disassembly of ectoplasmic specializations in the rat testis.
Ectoplasmic specializations are testis specific intercellular adhesion junctions found in Sertoli cells. They are tripartite structures consisting of the plasma membrane of the Sertoli cell, a submembrane layer of actin filaments and an attached cistern of endoplasmic reticulum. Ectoplasmic specializations occur in areas of attachment to spermatids and as part of the basal junction complex between neighboring Sertoli cells. They are functionally related to a number of fundamental events that occur during spermatogenesis, including attachment and then release of developing sperm cells and the translocation of spermatocytes through the blood-testis barrier. The structures may contain viable molecular targets for the development of contraceptives. Here we describe techniques for obtaining, from rat testes, testicular fractions enriched for spermatids with attached ectoplasmic specializations and for disassembling the complexes with gelsolin to obtain supernatants enriched for plaque components. The techniques involve stripping the epithelium from tubule walls, mechanically fragmenting the epithelium, using step sucrose gradients to enrich for spermatids with attached junction plaques, and then incubating with exogenous gelsolin to release plaque components into solution.
32.Guttman, JA; Kazemi, P; Lin, AE; Vogl, AW; Finlay, BB. (2007) Desmosomes are unaltered during infections by attaching and effacing pathogens.Anatomical Record-Advances in Integrative Anatomy and Evolutionary Biology 290: 199-205 Desmosomes are unaltered during infections by attaching and effacing pathogens
EPEC; citrobacter rodentium; desmosome; junction; pathogen ENTEROPATHOGENIC ESCHERICHIA-COLI; SCALDED-SKIN SYNDROME; DISRUPTION IN-VIVO; CITROBACTER-RODENTIUM; MDCK CELLS; BARRIER FUNCTION; TIGHT JUNCTIONS; PROTEIN; MICE; ESPG
The human attaching and effacing (A/E) intestinal pathogens enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), and the murine A/E pathogen Citrobacter rodentium cause serious diarrhea in their hosts. These bacteria alter numerous host cell components, including organelles, the host cell cytoskeleton, and tight junctions during the infectious process. One of the proteins that contribute to the intermediate filament network in host cells, cytokeratin-18, is extensively altered during EPEC infections. Based on this, we tested the hypothesis that desmosomes, the only intercellular junctions that interact with intermediate filaments, are also influenced by A/E pathogen infections. We found that the desmosomal transmembrane proteins desmoglein and desmocollin, as well as the desmosome plaque protein desmoplakin, all remain unchanged during EPEC infection in vitro. This evidence is corroborated by the unaltered localization of desmoglein and desmoplakin in vivo in mice infected with C. rodentium for 7 days. Electron microscopic analysis of 7-day C. rodentium-infected murine colonocytes also show no observable differences in the desmosomes when compared to uninfected controls. Our data suggest that, unlike tight junctions, the desmosome protein levels and localization, as well as desmosome morphology, are unaltered during A/E pathogenesis. DOI
30.Guttman, JA; Vaid, KS; Vogl, AW. (2007) A re-evaluation of gelsolin at ectoplasmic specializations in sertoli cells: The influence of serum in blocking buffers on staining patterns.Anatomical Record-Advances in Integrative Anatomy and Evolutionary Biology 290: 324-329 A re-evaluation of gelsolin at ectoplasmic specializations in sertoli cells: The influence of serum in blocking buffers on staining patterns
gelsolin; ectoplasmic specializations; serum; testis; Sertoli
In this study, we test the hypothesis that gelsolin immunolocalized in actin filament-rich ectoplasmic specializations may be exogenous gelsolin present in normal serum used in blocking buffers, and that binds to the intercellular adhesion plaques during tissue processing. Fixed frozen sections of rat and rabbit testis were pre-treated with standard blocking buffers containing 5% normal goat serum (NGS) and then incubated with anti-gelsolin antibodies in the presence of 1% NGS. Other sections were treated in a similar fashion, but in buffers not containing NGS. Sections were then labeled with secondary antibody conjugated to a fluorochrome. Localized staining at ectoplasmic specializations occurred only in sections treated with NGS. The only positive staining in sections not treated with NGS was associated with seminiferous tubule walls and blood vessels in rabbit tissue. The antibodies reacted with a single band at the appropriate molecular weight for gelsolin on immunoblots of NGS, but did not react on immunoblots of testis or seminiferous epithelium. We conclude that gelsolin localized at ectoplasmic specializations using current commercially available antibodies is a result of non-specific binding to the fixed tissues of gelsolin present in blocking buffers. DOI
28. Vaid, KS; Guttman, JA; Babyak, N; Deng, W; McNiven, MA; Mochizuki, N; Finlay, BB; Vogl, AW. (2007) The role of dynamin 3 in the testis.Journal of Cellular Physiology 210: 644-654 The role of dynamin 3 in the testis
We report here that dynamin 3 in the testis is associated with structures termed tubulobulbar complexes that internalize intact intercellular junctions during sperm release and turnover of the blood-testis barrier. The protein lies adjacent to an actin-Arp2/3 network that cuffs the double plasma membrane tubular invagination at the core of each complex. To explore the possible relationship between dynamin 3 and nectin-based adhesion junctions, we transiently transfected DsRed-tagged dynamin 3 into MDCK cells stably transfected with eGFP-tagged nectin 2, one of the adhesion molecules known to be expressed in Sertoli cells at adhesion junctions. Cells transfected with the dynamin 3 construct had less uniformly distributed nectin 2 at intercellular contacts when compared to control cells expressing only nectin 2 or transfected with the DsRed plasmid alone. Significantly, tubular extensions positive for nectin 2 were visible projecting into the cells from regions of intercellular contact. Our findings are consistent with the conclusion that dynamin 3 is involved with tubulobulbar morphogenesis. Dynamin 3 also occurs in concentrated deposits around the capitulum and striated columns in the connecting piece of sperm tails suggesting that the protein in these cells may function to stabilize the base of the tail or serve as a reservoir for use during or after fertilization. DOI
27. Vaid, KS; Guttman, JA; Singaraja, RR; Vogl, AW. (2007) A kinesin is present at unique sertoli/spermatid adherens junctions in rat and mouse testes.Biology of Reproduction 77: 1037-1048 A kinesin is present at unique sertoli/spermatid adherens junctions in rat and mouse testes
adherens junctions; ectoplasmic specializations; kinesin; Sertoli cells; spermatid translocation; testis
During spermatogenesis, spermatids undergo a "down and up" translocation event in the seminiferous epithelium. This event has been proposed to result from the movement of ectoplasmic specializations, which are formed in Sertoli cells at sites of adhesion to spermatids, along adjacent microtubule tracts. To test the hypothesis that a kinesin is associated with ectoplasmic specializations, we generated antibodies to conserved kinesin sequences and detected kinesins on fixed frozen testis sections and fixed seminiferous epithelial fragments. The antibodies reacted with ectoplasmic specializations related to spermatids, in addition to reacting with other structures in the epithelium known to contain kinesins. At the electron microscopy level, the antibodies reacted with the cytoplasmic face of the endoplasmic reticulum component of ectoplasmic specializations. Based on mRNA transcript screens using mouse GeneChip arrays of testis and Sertoli cells, we identified KIF20 as a candidate kinesin at ectoplasmic specializations. Antibodies generated against a peptide sequence unique to this kinesin reacted at ectoplasmic specializations in testis sections and epithelial fragments, as well as with the endoplasmic reticulum component of ectoplasmic specializations when analyzed by electron microscopy. The antibody reacted on Western blots with full-length KIF20. On Western blots of testis lysates, the antibody reacted with a protein that is not present in other tissues and which migrates at a higher molecular weight than that predicted for KIF20. Our results demonstrate that a kinesin is associated with apical ectoplasmic specializations in Sertoli cells and that the motor may be an isoform of KIF20. DOI
26. Veiga E, Guttman JA, Bonazzi M, Boucrot E, Toledo-Arana A, Lin AE, Enninga J, Pizarro-Cerd√° J, Finlay BB, Kirchhausen T, Cossart P. (2007) Invasive and adherent bacterial pathogens co-Opt host clathrin for infection.Cell Host & Microbe 2(5):340-51. Invasive and adherent bacterial pathogens co-Opt host clathrin for infection.
Infection by the bacterium Listeria monocytogenes depends on host cell clathrin. To determine whether this requirement is widespread, we analyzed infection models using diverse bacteria. We demonstrated that bacteria that enter cells following binding to cellular receptors (termed zippering bacteria) invade ina clathrin-dependent manner. In contrast, bacteria that inject effector proteins into host cells in order to gain entry (termed triggering bacteria) invade in a clathrin-independent manner. Strikingly, enteropathogenic Escherichia coli (EPEC) required clathrin to form actin-rich pedestals in host cells beneath adhering bacteria, even though this pathogen remains extracellular. Furthermore, clathrin accumulation preceded the actin rearrangements necessary for Listeria entry. These data provide evidence fora clathrin-based entry pathway allowing internalization of large objects (bacteria and ligand-coated beads) and used by zippering bacteria as part of a general mechanism to invade host mammalian cells. We also revealed a nonendocytic role for clathrin required for extracellular EPEC infections.Website DOI
24.Guttman, JA; Li, YL; Wickham, ME; Deng, WY; Vogl, AW; Finlay, BB. (2006) Attaching and effacing pathogen-induced tight junction disruption in vivo.Cellular Microbiology 8: 634-645 Attaching and effacing pathogen-induced tight junction disruption in vivo
Diarrhoea is a hallmark of infections by the human attaching and effacing (A/E) pathogens, enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). Although the mechanisms underlying diarrhoea induced by these pathogens remain unknown, cell culture results have suggested that these pathogens may target tight junctions. Tight junctions in the colon function as physical intercellular barriers that separate and prevent mixing of the luminal contents with adlumenal regions of the epithelium. Consequently, it is thought that the disruption of intestinal epithelial tight junctions by A/E pathogens could result in a loss of barrier function in the alimentary tract; however, this remains unexamined. Here we demonstrate for the first time that A/E pathogen infection results in the morphological alteration of tight junctions during natural disease. Tight junction alteration, characterized by relocalization of the transmembrane tight junction proteins claudin 1, 3 and 5, is a functional disruption; molecular tracers, which do not normally penetrate uninfected epithelia, pass across pathogen-infected epithelia. Functional junction disruption occurs with a concomitant increase in colon luminal water content. The effects on tissue are dependent upon the bacterial type III effector EspF (E. coli secreted protein F), because bacteria lacking EspF, while able to colonize, are defective for junction disruption and result in decreased proportions of water in the colon compared with wild-type infection. These results suggest that the diarrhoea induced by A/E pathogens occurs as part of functional tight junction disruption. DOI
22.Guttman, JA; Samji, FN; Li, YL; Vogl, AW; Finlay, BB. (2006) Evidence that tight junctions are disrupted due to intimate bacterial contact and not inflammation during attaching and effacing pathogen infection in vivo.Infection and Immunity 74: 6075-6084 Evidence that tight junctions are disrupted due to intimate bacterial contact and not inflammation during attaching and effacing pathogen infection in vivo
It is widely accepted that tight junctions are altered during infections by attaching and effacing (A/E) pathogens. These disruptions have been demonstrated both in vitro and more recently in vivo. For in vivo experiments, the murine model of A/E infection with Citrobacter rodentium is the animal model of choice. In addition to effects on tight junctions, these bacteria also colonize the colon at high levels, efface colonocyte microvilli, and cause hyperplasia and inflammation. Although we have recently demonstrated that tight junctions are disrupted by C. rodentium, the issue of direct effects of bacteria on epithelial cell junctions versus the indirect effects of inflammation still remains to be clarified. Here, we demonstrate that during the C. rodentium infections, inflammation plays no discernible role in the alteration of tight junctions. The distribu- tion of the tight junction proteins, claudin-1, -3, and -5, are unaffected in inflamed colon, and junctions appear morphologically unaltered when viewed by electron microscopy. Additionally, tracer molecules are not capable of penetrating the inflamed colonic epithelium of infected mice that have cleared the bacteria. Finally, infected colonocytes from mice exposed to C. rodentium for 14 days, which have high levels of bacterial attachment to colonocytes as well as inflammation, have characteristic, altered claudin localization whereas cells adjacent to infected colonocytes retain their normal claudin distribution. We conclude that inflammation plays no dis- cernible role in tight junction alteration during A/E pathogenesis and that tight junction disruption in vivo appears dependent only on the direct intimate attachment of the pathogenic bacteria to the cells. DOI
21. Ma, CX; Wickham, ME; Guttman, JA; Deng, WY; Walker, J; Madsen, KL; Jacobson, K; Vogl, WA; Finlay, BB; Vallance, BA. (2006) Citrobacter rodentium infection causes both mitochondrial dysfunction and intestinal epithelial barrier disruption in vivo: role of mitochondrial associated protein (Map).Cell Microbiol. 8: 1669-1686 Citrobacter rodentium infection causes both mitochondrial dysfunction and intestinal epithelial barrier disruption in vivo: role of mitochondrial associated protein (Map)
Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli are non-invasive attach- ing/effacing (A/E) bacterial pathogens that infect their host’s intestinal epithelium, causing severe diar- rhoeal disease. These bacteria utilize a type III secre- tion apparatus to deliver effector molecules into host cells, subverting cellular function. Mitochondrial associated protein (Map) is a multifunctional effector protein that targets host cell mitochondria and con- tributes to infection-induced epithelial barrier dys- function in vitro. Unfortunately, the relevance of these actions to the pathogenesis of EPEC-induced disease is uncertain. Using Citrobacter rodentium, a mouse- adapted A/E bacterium, we found that Map colocal- ized with host cell mitochondria, and that in vivo infection led to a disruption of mitochondrial mor- phology in infected colonocytes as assessed by electron microscopy. Histochemical staining for the mitochondrial enzyme succinate dehydrogenase also revealed a significant loss of mitochondrial respira- tory function in the infected intestinal epithelium; however, both pathologies were attenuated in mice infected with a Dmap strain. C. rodentium Map was also implicated in the disruption of epithelial barrier function both in vitro and in vivo. These studies thus advance our understanding of how A/E pathogens subvert host cell functions and cause disease, dem- onstrating that Map contributes to the functional disruption of the intestinal epithelium during enteric infection by C. rodentium. DOI
19.Guttman, JA; Obinata, T; Shima, J; Griswold, M; Vogl, AW. (2004) Non-muscle cofilin is a component of tubulobulbar complexes in the testis.Biology of Reproduction 70: 805-812 Non-muscle cofilin is a component of tubulobulbar complexes in the testis
Sertoli cells; spermatogenesis; testis
Tubulobulbar complexes are finger-like structures that form at the interface between maturing spermatids and Sertoli cells prior to sperm release and at the interface between two Sertoli cells near the base of the seminiferous epithelium. They originate in areas previously occupied by actin filament-associated intercellular adhesion plaques known as ectoplasmic specializations. Actin filaments also are associated with tubulobulbar complexes where they appear to form a network, rather than the tightly packed bundles found in ectoplasmic specializations. Cofilin, a calcium-independent actin-depolymerizing protein, previously has been identified in the testis, but has not been localized to specific structures in the seminiferous epithelium. To determine if cofilin is found in Sertoli cells and is concentrated at actin-rich structures, we reacted fixed frozen sections of rat testis, fixed fragmented tissue, and blots of seminiferous epithelium with pan-specific and non-muscle cofilin antibodies. in addition, GeneChip microarrays (Affymetrix, Santa Clara, CA) were utilized to determine the abundance of mRNA for all cofilin isoforms in Sertoli cells. Using the monoclonal pan-specific cofilin antibody, we found specific labeling exclusively at tubulobulbar complexes and not at ectoplasmic specializations. On one-dimensional (1D) Western blots this antibody reacted monospecifically with one band, and on 2D blots reacted with two dots, which we interpret as phosphorylated and nonphosphorylated forms of a single cofilin isotype. Messenger RNA for nonmuscle cofilin in Sertoli cells is about 8.5-fold higher than for muscle-type cofilin. To confirm that the non-muscle isoform of cofilin is present at tubulobulbar complexes, we used antibodies specific to non-muscle cofilin for immunofluorescent localization. As with the pan-specific antibody, we found that the nonmuscle cofilin antibody exclusively labeled tubulobulbar complexes. Results presented here indicate that non-muscle cofilin is concentrated at tubulobulbar complexes. Our results also indicate that cofilin is not concentrated at ectoplasmic specializations. DOI
18.Guttman, JA; Takai, Y; Vogl, AW. (2004) Evidence that tubulobulbar complexes in the seminiferous epithelium are involved with internalization of adhesion junctions.Biology of Reproduction 71: 548-559 Evidence that tubulobulbar complexes in the seminiferous epithelium are involved with internalization of adhesion junctions
Sertoli cells; spermatid; spermatogenesis; testis
Tubulobulbar complexes may be part of the mechanism by which intercellular adhesion junctions are internalized by Sertoli cells during sperm release. These complexes develop in regions where Sertoli cells are attached to adjacent cells by intercellular adhesion junctions termed ectoplasmic specializations. At sites where Sertoli cells are attached to spermatid heads, tubulobulbar complexes consist of fingerlike processes of the spermatid plasma membrane, corresponding invaginations of the Sertoli cell plasma membrane, and a surrounding cuff of modified Sertoli cell cytoplasm. At the terminal ends of the complexes occur clusters of vesicles. Here we show that tubulobulbar complexes develop in regions previously occupied by ectoplasmic specializations and that the structures share similar molecular components. In addition, the adhesion molecules nectin 2 and nectin 3, found in the Sertoli cell and spermatid plasma membranes, respectively, are concentrated at the distal ends of tubulobulbar complexes. We also demonstrate that double membrane bounded vesicles are associated with the ends of tubulobulbar complexes and nectin 3 is present on spermatids, but is absent from spermatozoa released from the epithelium. These results are consistent with the conclusion that Sertoli cell and spermatid membrane adhesion domains are internalized together by tubulobulbar complexes. PKCalpha, a kinase associated with endocytosis of adhesion domains in other systems, is concentrated at tubulobulbar complexes, and antibodies to endosomal and lysosomal (LAMP1, SGP1) markers label the cluster of vesicles associated with the ends of tubulobulbar complexes. Our results are consistent with the conclusion that tubulobulbar complexes are involved with the disassembly of ectoplasmic specializations and with the internalization of intercellular membrane adhesion domains during sperm release. DOI
15.Guttman, JA; Janmey, P; Vogl, AW. (2002) Gelsolin - evidence for a role in turnover of junction-related actin filaments in Sertoli cells.Journal of Cell Science 115: 499-505 Gelsolin - evidence for a role in turnover of junction-related actin filaments in Sertoli cells
gelsolin; adhesion junctions; ectoplasmic specializations
The gelsolin-phosphoinositide pathway may be part of the normal mechanism by which Sertoli cells regulate sperm release and turnover of the blood-testis barrier. The intercellular adhesion complexes (ectoplasmic specializations) involved with these two processes are tripartite structures consisting of the plasma membrane, a layer of actin filaments and a cistern of endoplasmic reticulum. Gelsolin is concentrated in these adhesion complexes. In addition, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P-2) and phosphoinositide-specific phospholipase C are found in the structures. Treatment of isolated spermatid/junction complexes with exogenous phosphoinositide-specific phospholipase Q or with a synthetic peptide consisting of the PtdIns(4,5)P-2 binding region of gelsolin, results in the release of gelsolin and loss of actin from the adhesion complexes. We present a model for the disassembly of the actin layer of the adhesion complex that involves the hydrolysis of PtdIns(4,5)P-2 resulting in the release of gelsolin within the plaque. Further, we speculate that the hydrolysis of PtdIns(4,5)P-2 may result in a local Ca2+ surge via the action of inositol. triphosphate on junctional endoplasmic reticulum. This Ca2+ surge would facilitate the actin severing function of gelsolin within the adhesion complex.Website
12. Velichkova, M; Guttman, J; Warren, C; Eng, L; Kline, K; Vogl, AW; Hasson, T. (2002) A human homologue of Drosophila Kelch associates with myosin-VIIa in speciaiized adhesion junctions.Cell Motility and the Cytoskeleton 51: 147-164 A human homologue of Drosophila Kelch associates with myosin-VIIa in speciaiized adhesion junctions
unconventional myosin; actin; focal adhesion plaque; Kelch protein; SH3 domain
Mutations in myosin-VIIa are responsible for the deaf-blindness, Usher disease. Myosin-VIIa is also highly expressed in testis, where it is associated with specialized adhesion plaques termed ectoplasmic specializations (ES) that form between Sertoli cells and germ cells. To identify new roles for myosin-VIIa, we undertook a yeast two-hybrid screen to identify proteins associated with myosin-VIIa in the ES. We identified Keap1, a human homologue of the Drosophila ring canal protein, kelch. The kelch-repeats in the C-terminus of human Keap1 associate with the SH3 domain of myosin-VIIa. Immunolocalization studies revealed that Keap1 is present with myosin-VIIa in the actin bundles of the ES. Myosin-VIIa and Keap1 copurify with ES and colocate with each other and with F-actin at the electron microscopy level. Interestingly, in many epithelial cell types including cells derived from retina and inner ear, Keap1 is a component of focal adhesions and zipper junctions. Keap1 can target to the ES in the absence of myosin-VIIa, suggesting that Keap1 associates with other molecules in the adhesion plaque. Keap1 and myosin-VIIa overlapped in expression in the inner hair cells of the cochlea, suggesting that Keap1 may be a part of a family of actin-binding proteins that could be important for myosin-VIIa function in testis and inner ear. (C) 2002 Wiley-Liss, Inc. DOI
9. Leavitt, BR; Guttman, JA; Hodgson, JG; Kimel, GH; Singaraja, R; Vogl, AW; Hayden, MR. (2001) Wild-type huntingtin reduces the cellular toxicity of mutant huntingtin in vivo.American Journal of Human Genetics 68: 313-324 Wild-type huntingtin reduces the cellular toxicity of mutant huntingtin in vivo
We have developed yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 or YAC72) human huntingtin (htt), in a developmental- and tissue-specific manner, that is identical to endogenous htt. YAC72 mice develop selective degeneration of medium spiny projection neurons in the lateral striatum, similar to what is observed in Huntington disease. Mutant human htt expressed by YAC transgenes can compensate for the absence of endogenous htt and can rescue the embryonic lethality that characterizes mice homozygous for targeted disruption of the endogenous Hdh gene (-/-). YAC72 mice lacking endogenous htt (YAC72 -/-) manifest a novel phenotype characterized by infertility, testicular atrophy, aspermia, and massive apoptotic cell death in the testes. The testicular cell death in YAC72 -/- mice can be markedly reduced by increasing endogenous htt levels. YAC72 mice with equivalent levels of both wild-type and mutant htt (YAC72 +/+) breed normally and have no evidence of increased testicular cell death. Similar findings are seen in YAC46 -/- mice compared with YAC46 +/+ mice, in which wild-type htt can completely counteract the proapoptotic effects of mutant htt. YAC18 -/- mice display no evidence of increased cellular apoptosis, even in the complete absence of endogenous htt, demonstrating that the massive cellular apoptosis observed in YAC46 -/- mice and YAC72 -/- mice is polyglutamine-mediated toxicity from the mutant transgene. These data provide the first direct in vivo evidence of a role for wild-type htt in decreasing the cellular toxicity of mutant htt. DOI
7.Guttman, JA; Kimel, GH; Vogl, AW. (2000) Dynein and plus-end microtubule-dependent motors are associated with specialized Sertoli cell junction plaques (ectoplasmic specializations).Journal of Cell Science 113: 2167-2176 Dynein and plus-end microtubule-dependent motors are associated with specialized Sertoli cell junction plaques (ectoplasmic specializations)
dynein; kinesin; ectoplasmic specialization; Sertoli cell
The mechanism responsible for spermatid translocation in the mammalian seminiferous epithelium was proposed to be the microtubule-based transport of specialized junction plaques (ectoplasmic specializations) that occur in Sertoli cell regions attached to spermatid heads. These plaques each consist of a cistern of endoplasmic reticulum, a layer of actin filaments and the adjacent plasma membrane. It is predicted that motor proteins function to move the junction plaques, and hence the attached spermatids, first towards the base and then back to the apex of the epithelium, along microtubules. If this hypothesis is true, motor proteins should be associated with the cytoplasmic face of the endoplasmic reticulum component of ectoplasmic specializations. In addition, isolated junction plaques should support microtubule movement both in the plus and minus directions to account for the bidirectional translocation of spermatids in vivo. To determine if cytoplasmic dynein is localized to the endoplasmic reticulum of the plaques, perfusion-fixed rat testes were Immunologically probed, at the ultrastructural level, for the intermediate chain of cytoplasmic dynein (IC74). In addition, testicular fractions enriched for spermatid/junction complexes were incubated with and without gelsolin, centrifuged and the supernatants compared, by western blot analysis, for Glucose Regulated Protein 94 (a marker for endoplasmic reticulum) and IC74. At the ultrastructural level, the probe for IC74 clearly labelled material associated with the cytoplasmic face of the endoplasmic reticulum component of the junction plaques. In the gelsolin experiments, both probes reacted more strongly with appropriate bands from the gelsolin-treated supernatants than with corresponding bands from controls. To determine if the junction plaques support microtubule transport in both directions, polarity-labelled microtubules were bound to isolated spermatid/junction complexes and then assessed for motility in the presence of ATP and testicular cytosol (2 mg/ml). Of 25 recorded motility events, 17 were in a direction consistent with a plus-end directed motor being present, and 8 were in the minus-end direction. The results are consistent with the conclusion that the junction plaques have the potential for moving along microtubules in both the plus and minus directions and that both a kinesin-type and a dynein-type motor may be associated with the junction plaques. The data also indicate that cytoplasmic dynein is localized to the cytoplasmic face of the endoplasmic reticulum component of the plaques.Website
4. Vogl, AW; Pfeiffer, DC; Mulholland, D; Kimel, G; Guttman, J. (2000) Unique and multifunctional adhesion junctions in the testis: Ectoplasmic specializations.Archives of Histology and Cytology 63: 1-15 Unique and multifunctional adhesion junctions in the testis: Ectoplasmic specializations
In this paper, we review the structure and function of a unique type of actin-related intercellular adhesion junctions in the testis, Based on their ultrastructure, the junctions are divided into five distinct domains, The currently identified molecular components of each domain are summarized. In addition, the architecture of the mammalian system is compared with that of non-mammalian vertebrates. Functionally, the junctions are related to the turnover of adhesion between Sertoli cells, to the attachment of spermatids to the seminiferous epithelium, and to sperm release. They also are part of the mechanism by which spermatids are moved through the epithelium. Evidence consistent with adhesion and motility related functions is discussed. Control, both of junction turnover and of microtubule-based transport, is identified as an important avenue for future research. DOI
3.Guttman, JA; Mulholland, DJ; Vogl, AW. (1999) Plectin is concentrated at intercellular junctions and at the nuclear surface in morphologically differentiated rat Sertoli cells.Anatomical Record 254: 418-428 Plectin is concentrated at intercellular junctions and at the nuclear surface in morphologically differentiated rat Sertoli cells
Sertoli cells; plectin; cytoskeleton
Intermediate filaments in Sertoli cells have a well-defined pattern of distribution. They form a basally situated perinuclear network from which filaments extend peripherally to adhesion plaques at the plasma membrane and to sites of codistribution with other major elements of the cytoskeleton, particularly with microtubules, Although the general pattern of intermediate filament distribution is known, the molecular components involved with linking the filaments to organelles and attachment plaques in these cells have not been identified. One candidate for such a linking element is plectin. In this study we test for the presence of, and determine the distribution of, plectin in Sertoli cells of the rat testis. Fixed frozen sections and fixed epithelial fragments of rat testis were probed far plectin and vimentin using antibodies; Tissue-was evaluated using standard fluorescence microscopy and confocal microscopy. Plectin in Sertoli cells was concentrated in a narrow zone surrounding the nucleus, and at focal sites, presumably desmosome-like plaques, at interfaces with adjacent cells. Plectin was also concentrated at sites where? intermediate filament bundles project into specialized actin-filament containing plaques at sites of attachment to elongate spermatids. Plectin in Sertoli cells is concentrated at the nuclear surface and in junction plaques associated with the plasma membrane. The pattern of distribution is consistent with plectin being involved with linking intermediate filaments centrally (basally) to; the nucleus and peripherally to intercellular attachment sites. Anat Rec 254:418-428, 1999. (C) 1999 Wiley-Liss, Inc. DOI
