scholarly journals Palladin Is a Regulator of Actin Filament Bundles at the Ectoplasmic Specialization in Adult Rat Testes

Endocrinology ◽  
2013 ◽  
Vol 154 (5) ◽  
pp. 1907-1920 ◽  
Author(s):  
Xiaojing Qian ◽  
Dolores D. Mruk ◽  
Elissa W. P. Wong ◽  
Pearl P. Y. Lie ◽  
C. Yan Cheng
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Plasma Membranes ◽  
Permeability Barrier ◽  
Protein Distribution ◽  
Filament Bundles ◽  
Ectoplasmic Specialization ◽  
Blood Testis Barrier

Abstract In rat testes, the ectoplasmic specialization (ES) at the Sertoli-Sertoli and Sertoli-spermatid interface known as the basal ES at the blood-testis barrier and the apical ES in the adluminal compartment, respectively, is a testis-specific adherens junction. The remarkable ultrastructural feature of the ES is the actin filament bundles that sandwiched in between the cisternae of endoplasmic reticulum and apposing plasma membranes. Although these actin filament bundles undergo extensive reorganization to switch between their bundled and debundled state to facilitate blood-testis barrier restructuring and spermatid adhesion/transport, the regulatory molecules underlying these events remain unknown. Herein we report findings of an actin filament cross-linking/bundling protein palladin, which displayed restrictive spatiotemporal expression at the apical and the basal ES during the epithelial cycle. Palladin structurally interacted and colocalized with Eps8 (epidermal growth factor receptor pathway substrate 8, an actin barbed end capping and bundling protein) and Arp3 (actin related protein 3, which together with Arp2 form the Arp2/3 complex to induce branched actin nucleation, converting bundled actin filaments to an unbundled/branched network), illustrating its role in regulating actin filament bundle dynamics at the ES. A knockdown of palladin in Sertoli cells in vitro with an established tight junction (TJ)-permeability barrier was found to disrupt the TJ function, which was associated with a disorganization of actin filaments that affected protein distribution at the TJ. Its knockdown in vivo also perturbed F-actin organization that led to a loss of spermatid polarity and adhesion, causing defects in spermatid transport and spermiation. In summary, palladin is an actin filament regulator at the ES.

scholarly journals rpS6 Regulates Blood-Testis Barrier Dynamics By Affecting F-Actin Organization and Protein Recruitment

Endocrinology ◽  
2012 ◽  
Vol 153 (10) ◽  
pp. 5036-5048 ◽  
Author(s):  
Ka-Wai Mok ◽  
Dolores D. Mruk ◽  
Bruno Silvestrini ◽  
C. Yan Cheng
Keyword(s):  
Sertoli Cell ◽  
Actin Filament ◽  
Sertoli Cells ◽  
Plasma Membranes ◽  
Permeability Barrier ◽  
Actin Organization ◽  
Filament Bundles ◽  
Mtorc1 Pathway ◽  
Protein Recruitment ◽  
Blood Testis Barrier

Abstract During spermatogenesis, preleptotene spermatocytes residing near the basement membrane of the seminiferous tubule must traverse the blood-testis barrier (BTB) at stage VIII–IX of the epithelial cycle to continue their development in the adluminal compartment. Unlike other blood-tissue barriers (e.g. the blood-brain barrier) that are created by the endothelial tight junction (TJ) barrier of capillaries, the BTB is created by specialized junctions between Sertoli cells in which TJ coexists with basal ectoplasmic specialization (basal ES, a testis-specific adherens junction). The basal ES is typified by the presence of tightly packed actin filament bundles sandwiched between cisternae of endoplasmic reticulum and the apposing plasma membranes of Sertoli cells. These actin filament bundles also confer unusual adhesive strength to the BTB. Yet the mechanisms by which these filamentous actin (F-actin) networks are regulated from the bundled to the debundled state to facilitate the transit of spermatocytes remain elusive. Herein, we provide evidence that ribosomal protein S6 (rpS6), the downstream signaling molecule of the mammalian target of rapamycin complex 1 (mTORC1) pathway, is a major regulator of F-actin organization and adhesion protein recruitment at the BTB. rpS6 is restrictively and spatiotemporally activated at the BTB during the epithelial cycle. An activation of rpS6 led to a disruption of the Sertoli cell TJ barrier and BTB integrity. Its silencing in vitro or in vivo by using small interfering RNA duplexes or short hairpin RNA was found to promote the Sertoli cell TJ permeability barrier by the recruitment of adhesion proteins (e.g. claudin-11 and occludin) to the BTB. Thus, rpS6 in the mTORC1 pathway regulates BTB restructuring via its effects on the F-actin organization and protein recruitment at the BTB.

scholarly journals An Occludin-Focal Adhesion Kinase Protein Complex at the Blood-Testis Barrier: A Study Using the Cadmium Model

Endocrinology ◽  
2009 ◽  
Vol 150 (7) ◽  
pp. 3336-3344 ◽  
Author(s):  
Erica R. Siu ◽  
Elissa W. P. Wong ◽  
Dolores D. Mruk ◽  
K. L. Sze ◽  
Catarina S. Porto ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Sertoli Cell ◽  
Focal Adhesion ◽  
Adhesion Molecule ◽  
Protein Complex ◽  
Permeability Barrier ◽  
Ectoplasmic Specialization ◽  
Blood Testis Barrier

Several integral membrane proteins that constitute the blood-testis barrier (BTB) in mammalian testes, in particular rodents, are known to date. These include tight junction (TJ) proteins (e.g. occludin, junctional adhesion molecule-A, claudins), basal ectoplasmic specialization proteins (e.g. N-cadherin), and gap junction proteins (e.g. connexin43). However, the regulators (e.g. protein kinases and phosphatases) that affect these proteins, such as their interaction with the cytoskeletal actin, which in turn confer cell adhesion at the TJ, remain largely unknown. We report herein that focal adhesion kinase (FAK) is a putative interacting partner of occludin, but not claudin-11 or junctional adhesion molecule-A. Immunohistochemistry and fluorescence microscopy studies illustrated that the expression of FAK in the seminiferous epithelium of adult rat testes was stage specific. FAK colocalized with occludin at the BTB in virtually all stages of the seminiferous epithelial cycle but considerably diminished in stages VIII–IX, at the time of BTB restructuring to facilitate the transit of primary leptotene spermatocytes. Using Sertoli cells cultured in vitro with established TJ-permeability barrier and ultrastructures of TJ, basal ectoplasmic specialization and desmosome-like junction that mimicked the BTB in vivo, FAK was shown to colocalize with occludin and zonula occludens-1 (ZO-1) at the Sertoli-Sertoli cell interface. When these Sertoli cell cultures were treated with CdCl2 to perturb the TJ-barrier function, occludin underwent endocytic-mediated internalization in parallel with FAK and ZO-1. Thus, these findings demonstrate that FAK is an integrated regulatory component of the occludin-ZO-1 protein complex, suggesting that functional studies can be performed to study the role of FAK in BTB dynamics.

scholarly journals Filamin A Is a Regulator of Blood-Testis Barrier Assembly during Postnatal Development in the Rat Testis

Endocrinology ◽  
2012 ◽  
Vol 153 (10) ◽  
pp. 5023-5035 ◽  
Author(s):  
Wenhui Su ◽  
Dolores D. Mruk ◽  
Pearl P. Y. Lie ◽  
Wing-yee Lui ◽  
C. Yan Cheng
Keyword(s):  
Postnatal Development ◽  
Sertoli Cell ◽  
Actin Filament ◽  
Permeability Barrier ◽  
Filamin A ◽  
Rat Testis ◽  
Filament Network ◽  
Blood Testis Barrier

Abstract The blood-testis barrier (BTB) is an important ultrastructure in the testis. A delay in its assembly during postnatal development leads to meiotic arrest. Also, a disruption of the BTB by toxicants in adult rats leads to a failure in spermatogonial differentiation. However, the regulation of BTB assembly remains unknown. Herein, filamin A, an actin filament cross-linker that is known to maintain and regulate cytoskeleton structure and function in other epithelia, was shown to be highly expressed during the assembly of Sertoli cell BTB in vitro and postnatal development of BTB in vivo, perhaps being used to maintain the actin filament network at the BTB. A knockdown of filamin A by RNA interference was found to partially perturb the Sertoli cell tight junction (TJ) permeability barrier both in vitro and in vivo. Interestingly, this down-regulating effect on the TJ barrier function after the knockdown of filamin A was associated with a mis-localization of both TJ and basal ectoplasmic specialization proteins. Filamin A knockdown also induced a disorganization of the actin filament network in Sertoli cells in vitro and in vivo. Collectively, these findings illustrate that filamin A regulates BTB assembly by recruiting these proteins to the microenvironment in the seminiferous epithelium to serve as the building blocks. In short, filamin A participates in BTB assembly by regulating protein recruitment during postnatal development in the rat testis.

scholarly journals Activation of the Arp2/3 Complex by the Actin Filament Binding Protein Abp1p

2001 ◽  
Vol 153 (3) ◽  
pp. 627-634 ◽  
Author(s):  
Bruce L. Goode ◽  
Avital A. Rodal ◽  
Georjana Barnes ◽  
David G. Drubin
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Mammalian Cells ◽  
Actin Assembly ◽  
Related Protein ◽  
Genetic Studies ◽  
Actin Networks ◽  
Specific Step

The actin-related protein (Arp) 2/3 complex plays a central role in assembly of actin networks. Because distinct actin-based structures mediate diverse processes, many proteins are likely to make spatially and temporally regulated interactions with the Arp2/3 complex. We have isolated a new activator, Abp1p, which associates tightly with the yeast Arp2/3 complex. Abp1p contains two acidic sequences (DDW) similar to those found in SCAR/WASp proteins. We demonstrate that mutation of these sequences abolishes Arp2/3 complex activation in vitro. Genetic studies indicate that this activity is important for Abp1p functions in vivo. In contrast to SCAR/WASp proteins, Abp1p binds specifically to actin filaments, not monomers. Actin filament binding is mediated by the ADF/cofilin homology (ADF-H) domain of Abp1p and is required for Arp2/3 complex activation in vitro. We demonstrate that Abp1p recruits Arp2/3 complex to the sides of filaments, suggesting a novel mechanism of activation. Studies in yeast and mammalian cells indicate that Abp1p is involved functionally in endocytosis. Based on these results, we speculate that Abp1p may link Arp2/3-mediated actin assembly to a specific step in endocytosis.

scholarly journals Actin filaments elongate from their membrane-associated ends

1981 ◽  
Vol 90 (2) ◽  
pp. 485-494 ◽  
Author(s):  
LG Tilney ◽  
EM Bonder ◽  
DJ DeRosier
Keyword(s):  
Plasma Membrane ◽  
Actin Filament ◽  
Actin Filaments ◽  
The Other ◽  
Dense Material ◽  
Thin Sections ◽  
Vacuole Membrane ◽  
Filament Bundles ◽  
Filament Bundle

In limulus sperm an actin filament bundle 55 mum in length extends from the acrosomal vacuole membrane through a canal in the nucleus and then coils in a regular fashion around the base of the nucleus. The bundle expands systematically from 15 filaments near the acrosomal vacuole to 85 filaments at the basal end. Thin sections of sperm fixed during stages in spermatid maturation reveal that the filament bundle begins to assemble on dense material attached to the acrosomal vacuole membrane. In micrographs fo these early stages in maturation, short bundles are seen extending posteriorly from the dense material. The significance is that these short, developing bundles have about 85 filaments, suggesting that the 85-filament end of the bundle is assembled first. By using filament bundles isolated and incubated in vitro with G actin from muscle, we can determine the end "preferred" for addition of actin monomers during polymerization. The end that would be associated with the acrosomal vacuole membrane, a membrane destined to be continuous with the plasma membrane, is preferred about 10 times over the other, thicker end. Decoration of the newly polymerized portions of the filament bundle with subfragment 1 of myosin reveals that the arrowheads point away from the acrosomal vacuole membrane, as is true of other actin filament bundles attached to membranes. From these observations we conclude that the bundle is nucleated from the dense material associated with the acrosomal vacuole and that monomers are added to the membrane-associated end. As monomers are added at the dense material, the thick first-made end of the filament bundle is pushed down through the nucleus where, upon reaching the base of the nucleus, it coils up. Tapering is brought about by the capping of the peripheral filaments in the bundle.

scholarly journals Enhancement of Actin-depolymerizing Factor/Cofilin-dependent Actin Disassembly by Actin-interacting Protein 1 Is Required for Organized Actin Filament Assembly in the Caenorhabditis elegans Body Wall Muscle

2006 ◽  
Vol 17 (5) ◽  
pp. 2190-2199 ◽  
Author(s):  
Kurato Mohri ◽  
Kanako Ono ◽  
Robinson Yu ◽  
Sawako Yamashiro ◽  
Shoichiro Ono
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Muscle Cells ◽  
Actin Depolymerizing Factor ◽  
Interacting Protein ◽  
Cellular Processes ◽  
Cytoskeletal Dynamics ◽  
End Capping

Regulated disassembly of actin filaments is involved in several cellular processes that require dynamic rearrangement of the actin cytoskeleton. Actin-interacting protein (AIP) 1 specifically enhances disassembly of actin-depolymerizing factor (ADF)/cofilin-bound actin filaments. In vitro, AIP1 actively disassembles filaments, caps barbed ends, and binds to the side of filaments. However, how AIP1 functions in the cellular actin cytoskeletal dynamics is not understood. We compared biochemical and in vivo activities of mutant UNC-78 proteins and found that impaired activity of mutant UNC-78 proteins to enhance disassembly of ADF/cofilin-bound actin filaments is associated with inability to regulate striated organization of actin filaments in muscle cells. Six functionally important residues are present in the N-terminal β-propeller, whereas one residue is located in the C-terminal β-propeller, suggesting the presence of two separate sites for interaction with ADF/cofilin and actin. In vitro, these mutant UNC-78 proteins exhibited variable alterations in actin disassembly and/or barbed end-capping activities, suggesting that both activities are important for its in vivo function. These results indicate that the actin-regulating activity of AIP1 in cooperation with ADF/cofilin is essential for its in vivo function to regulate actin filament organization in muscle cells.

scholarly journals Fascin 1 is an actin filament-bundling protein that regulates ectoplasmic specialization dynamics in the rat testis

2014 ◽  
Vol 307 (9) ◽  
pp. E738-E753 ◽  
Author(s):  
N. Ece Gungor-Ordueri ◽  
Ciler Celik-Ozenci ◽  
C. Yan Cheng
Keyword(s):  
Mammalian Cells ◽  
Early Stage ◽  
Seminiferous Epithelium ◽  
Permeability Barrier ◽  
Actin Organization ◽  
Rat Testis ◽  
Ectoplasmic Specialization ◽  
Stage Viii

In the testis, spermatids are polarized cells, with their heads pointing toward the basement membrane during maturation. This polarity is crucial to pack the maximal number of spermatids in the seminiferous epithelium so that millions of sperms can be produced daily. A loss of spermatid polarity is detected after rodents are exposed to toxicants (e.g., cadmium) or nonhormonal male contraceptives (e.g., adjudin), which is associated with a disruption on the expression and/or localization of polarity proteins. In the rat testis, fascin 1, an actin-bundling protein found in mammalian cells, was expressed by Sertoli and germ cells. Fascin 1 was a component of the ectoplasmic specialization (ES), a testis-specific anchoring junction known to confer spermatid adhesion and polarity. Its expression in the seminiferous epithelium was stage specific. Fascin 1 was localized to the basal ES at the Sertoli cell-cell interface of the blood-testis barrier in all stages of the epithelial cycle, except it diminished considerably at late stage VIII. Fascin 1 was highly expressed at the apical ES at stage VII–early stage VIII and restricted to the step 19 spermatids. Its knockdown by RNAi that silenced fascin 1 by ∼70% in Sertoli cells cultured in vitro was found to perturb the tight junction-permeability barrier via a disruption of F-actin organization. Knockdown of fascin 1 in vivo by ∼60–70% induced defects in spermatid polarity, which was mediated by a mislocalization and/or downregulation of actin-bundling proteins Eps8 and palladin, thereby impeding F-actin organization and disrupting spermatid polarity. In summary, these findings provide insightful information on spermatid polarity regulation.

The capactins, a class of proteins that cap the ends of actin filaments

1982 ◽  
Vol 299 (1095) ◽  
pp. 263-273 ◽  
Keyword(s):  
Skeletal Muscle ◽  
Actin Filament ◽  
Actin Filaments ◽  
Muscle Cells ◽  
Preliminary Evidence ◽  
The Other ◽  
Cellular Regulation ◽  
Filament Assembly ◽  
Filament Bundles

A number of proteins that bind specifically to the barbed ends of actin filaments in a cytochalasin-like manner have been purified to various degrees from a variety of muscle and non-muscle cells and tissues. Preliminary evidence also indicates that proteins that interact with the pointed ends of filaments are present in skeletal muscle. Because of their ability to cap one or the other end of an actin filament, we have designated this class of proteins as the ‘capactins’. On the basis of their effect on actin filament assembly and interaction in vitro , we propose that the capactins play important roles in cellular regulation of actin-based cytoskeletal and contractile functions. Our finding that the disappearance of actin filament bundles in virally transformed fibroblasts can be correlated with an increase in capactin activity in the extracts of these cells is consistent with this hypothesis.

scholarly journals Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks.

1981 ◽  
Vol 91 (3) ◽  
pp. 695-705 ◽  
Author(s):  
J V Small
Keyword(s):  
Electron Microscopy ◽  
Actin Filament ◽  
Actin Filaments ◽  
Osmium Tetroxide ◽  
Cultured Cells ◽  
Leading Edge ◽  
Cultured Fibroblasts ◽  
Cell Biol ◽  
Filament Bundles

The ordered structure of the leading edge (lamellipodium) of cultured fibroblasts is readily revealed in cells extracted briefly in Triton X-100-glutaraldehyde mixtures, fixed further in glutaraldehyde, and then negatively stained for electron microscopy. By this procedure, the leading edge regions show a highly organised, three-dimensional network of actin filaments together with variable numbers of radiating actin filament bundles or microspikes. The use of Phalloidin after glutaraldehyde fixation resulted in a marginal improvement in filament order. Processing of the cytoskeletons though the additional steps generally employed for conventional electron microscopy resulted in a marked deterioration or complete disruption of the order of the actin filament networks. In contrast, the actin filaments of the stress fiber bundles were essentially unaffected. Thus, postfixation in osmium tetroxide (1% for 7 min at room temperature) transformed the networks to a reticulum of kinked fibers, resembling those produced by the exposure of muscle F-actin to OsO4 in vitro (P. Maupin-Szamier and T. D. Pollard. 1978. J. Cell Biol. 77:837--852). While limited exposure to OsO4 (0.2+ for 20 min at 0 degrees C) obviated this destruction, dehydration in acetone or ethanol, with or without post-osmication, caused a further and unavoidable disordering and aggregation of the meshwork filaments. The meshwork regions of the leading edge then showed a striking resemblance to the networks hitherto described in critical point-dried preparations of cultured cells. I conclude that much of the "microtrabecular lattice" described by Wolosewick and Porter (1979. J. Cell Biol. 82:114--139) in the latter preparations constitutes actin meshworks and actin filament arrays, with their associated components, that have been distorted and aggregated by the preparative procedures employed.

scholarly journals INF2-mediated actin filament reorganization confers intrinsic resilience to neuronal ischemic injury

2021 ◽  
Author(s):  
Barbara Calabrese ◽  
Steven Jones ◽  
Yoko Yamaguchi-Shiraishi ◽  
Michael J Lingelbach ◽  
Uri Manor ◽  
...  
Keyword(s):  
Actin Filament ◽  
Dendritic Spines ◽  
Glucose Deprivation ◽  
Receptor Activation ◽  
Cell Swelling ◽  
Filament Bundles ◽  
Activation Conditions ◽  
Neuronal Swelling

During early stages of ischemic brain injury, glutamate receptor hyperactivation mediates neuronal death via osmotic cell swelling. Here we show that ischemia and excess NMDA receptor activation, conditions that trigger neuronal swelling, cause actin filaments to undergo a rapid and extensive reorganization within the somatodendritic compartment. Normally, Factin is concentrated within dendritic spines, with relatively little Factin in the dendrite shaft. However, beginning <5 min after incubation of neurons with NMDA, Factin depolymerizes within dendritic spines and polymerizes into long, stable filament bundles within the dendrite shaft and soma. A similar actinification of the somatodendritic compartment occurs after oxygen/glucose deprivation in vitro, and in mouse brain after photothrombotic stroke in vivo. Following transient, sub-lethal NMDA exposure these actin changes spontaneously reverse within 1-2 hours. A combination of Na+, Cl-, water, and Ca2+ entry are all necessary, but not individually sufficient, for induction of actinification. Spine F-actin depolymerization is also required. Actinification is driven by activation of the Factin polymerization factor inverted formin2 (INF2). Silencing of INF2 renders neurons more vulnerable to NMDA induced membrane leakage and cell death, and formin inhibition markedly increases ischemic infarct severity in vivo. These results show that ischemia induced actin filament reorganization within the dendritic compartment is an intrinsic pro-survival response that protects neurons from death induced by swelling.

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