Actin binding proteins,actin cytoskeleton and spermatogenesis – Lesson from toxicant models

Actin cytoskeleton is crucial to support spermatogenesis in the mammalian testis. However, the molecular mechanism(s) underlying changes of actin cytoskeletal organization in response to cellular events that take place across the seminiferous epithelium (e.g., self-renewal of spermatogonial stem cells, germ cell differentiation, meosis, spermiogenesis, spermiation) at specific stages of the epithelial cycle of spermatogenesis remain largely unexplored. This, at least in part, is due to the lack of suitable study models to identify the crucial regulatory proteins and to investigate how these proteins work in concert to support actin dynamics. Much of the information on the role of actin binding proteins in the literature, namely the actin bundling proteins, actin nucleation proteins and motor proteins, are either findings based on genetic models or morphological analyses. While this information is helpful to delineate the function of these proteins to support spermatogenesis, they are not helpful to identify the regulatory signaling proteins, the signaling pathways and the cascade of events to modulate actin cytoskeleton dynamics. Recent studies based on the use of toxicant models, both in vitro and in vivo, however, have bridged this gap by identifying putative regulatory and signaling proteins of actin cytoskeleton. Herein, we summarize and critically evaluate these findings. We also provide a hypothetical model by which actin cytoskeletal dynamics in Sertoli cells are regulated, which in turn supports spermatid transport across the seminiferous epithelium, and at the blood-testis barrier (BTB) during the epithelial cycle of spermatogenesis.     

CD2AP inhibits metastasis in gastric cancer by promoting cellular adhesion and cytoskeleton assembly

Diffuse gastric cancer (DGC) is a lethal malignancy lacking effective systemic therapy. Among the most provocative recent results in DGC has been that the alter of the cellular cytoskeleton and intercellular adhesion. CD2-associated protein (CD2AP) is one of the critical proteins regulating cytoskeleton assembly and intercellular adhesion. However, no study has investigated the expression and biological significance of CD2AP in gastric cancer (GC) to date. Therefore, the aim of our study was to explore if the expression of CD2AP is associated with any clinical features of GC and to elucidate the underlying mechanism. Immunohistochemistry of 620 patient tissue samples indicated that the expression of CD2AP is downregulated in DGC. Moreover, a low CD2AP level was indicative of poor patient prognosis. In vitro, forced expression of CD2AP caused a significant decrease in the migration and invasion of GC cells, whereas depletion of CD2AP had the opposite effect. Immunofluorescence analysis indicated that CD2AP promoted cellular adhesion and influenced cell cytoskeleton assembly via interaction with the F-actin capping protein CAPZA1. Overall, the upregulation of CD2AP could attenuate GC metastasis, suggesting CD2AP as a novel biomarker for the prognosis and treatment of patients with GC.     

Differences in the abilities of human tau isoforms to promote microtubule assembly.

Three isoforms of human tau protein were compared for their abilities to induce microtubule assembly. The three isoforms, tau 3 (tau containing three microtubule-binding domains), tau 4 (tau containing four microtubule-binding domains) and tau 4L (tau containing four microtubule binding domains plus a 58-amino-acid insert near the N-terminus) were expressed in E. coli and purified using ammonium sulfate precipitation, ion exchange, and size exclusion chromatography. All three isoforms induced microtubule assembly at micromolar concentrations and showed similar critical concentrations for assembly of 0.4-0.45 microM. However, tau 4 induced microtubule formation at a rate five- to tenfold faster than either tau 3 or tau 4L. The rate of microtubule elongation seen with tau 4 was twofold greater than with tau 3 or tau 4L, suggesting that the faster rate of microtubule assembly seen with tau 4 was due, at least in part, to faster elongation. Tau 4 induced a greater number of microtubules to form at steady state than did tau 3 or tau 4L. The microtubules generated with each tau isoform had similar steady-state length distributions and were equally susceptible to cold-induced disassembly. These results indicate that the additional microtubule-binding domain in tau 4 enhances microtubule assembly, while the 58-amino-acid insert negates the stimulatory effect of the fourth microtubule-binding domain.     

Tau protein induces bundling of microtubules in vitro: comparison of different tau isoforms and a tau protein fragment.

Expression of tau protein in non-neuronal cells can result in a redistribution of the microtubule cytoskeleton into thick bundles of tau-containing microtubules (Lewis et al.: Nature 342:498-505, 1989; Kanai et al.: J Cell Biol 109:1173-1184, 1989). We reconstituted microtubule bundles using purified tubulin and tau in order to study the assembly of these structures. Taxol-stabilized tubulin polymers were incubated with various concentrations of recombinant human tau and examined by electron microscopy. With increasing concentrations of tau 3 (tau isoform containing three microtubule binding domains) or tau 4 (isoform containing four microtubule binding domains) the microtubules changed orientation from a random distribution to loosely and tightly packed parallel arrays and then to thick cables. In contrast, tau 4L, the tau isoform containing four microtubule binding domains plus a 58-amino acid insert near the N-terminus, showed minimal bundling activity. tau 4-induced bundling could be inhibited by the addition of 0.5 M NaCl or 0.4 mM estramustine phosphate, conditions which are known to inhibit tau binding to microtubules. A tau construct that contained only the microtubule binding domains plus 19 amino acids to the C-terminus was fully capable of bundling microtubules. Phosphorylation of tau 3 with cAMP-dependent protein kinase had no effect on its ability to induce microtubule bundling. These results indicate that tau protein is directly capable of bundling microtubules in vitro, and suggests that different tau isoforms differ in their ability to bundle microtubule filaments.     

Differential Distribution of MAP1a and Aldolase c in Adult Mouse Cerebellum

MAP1a is a microtubule-associated protein with an apparent molecular weight of 360 kDa that is found in the axonal and dendritic processes of neurons. Two monoclonal anti-MAP1a antibodies, anti-A and anti-BW6, revealed different epitope distributions in the adult mouse cerebellum. Anti-A stained Purkinje and granule cells uniformly throughout the cerebellum. In contrast, anti-BW6 selectively stained the dendrites of a subset of Purkinje cells, revealing parasagittal bands of immunoreactivity in the molecular layer. The compartmentation of the BW6 epitope was compared to the Purkinje cells as revealed by immunostaining with anti-zebrin II, a well known antigen expressed selectively by bands of Purkinje cells. The anti-BW6 staining pattern was complementary to the zebrin II bands, the zebrin II^- Purkinje cells having BW6⁺ dendrites. These results demonstrate that MAP1a is present in two forms in the mouse cerebellum, one of which is segregated into parasagittal bands. This may indicate a unique MAP1a isoform or may reflect differences in the metabolic states of Purkinje cell classes, and regional differences in their functions.     

Phosphorylation of neurofilament H subunit as related to arrangement of neurofilaments

To find out what causes differences in phosphorylation states in neurofilaments (NF), we selected two types of dendrite, one provided with very few NFs (Purkinje cell) and the other with relatively many (anterior horn cell). We examined these with four monoclonal antibodies selected by the Western blot analysis, two (NE14 and SMI31) recongnizing only phosphorylated, SMI32 recognizing only nonphosphorylated, and N52 recognizing phosphorylation-independent epitopes of NF-H. The immunoperoxidase labeling of dendrites, and also of perikarya, in both neurons was detectable with all four antibodies. After the tissue was treated with Triton X-100, the labeling was still detectable with SMI32 or N52, but undetectable with NE14 and SMI31. The brain homogenate Triton-extracted supernatant after centrifugation at 100,000g for 1 hr showed the staining of NE14, SMI31, and N52 but not that of SMI32. In Purkinje cell dendrite and perikaryon, NFs always appeared singly. In the immunogold labeling, they were labeled only with SMI32 or N52. Labeling by NE14 or SMI31 was distributed throughout the cytoplasm and hardly associated with NFs. In the anterior horn cell dendrite and perikaryon, NFs appeared both single and in bundles. They were predominantly labeled with SMI31 or N52 when they were single, and with NE14, SMI31, or N52 when they were bundled. Even in one NF, portions that appeared single were labeled mostly with SMI32 or N52, while the remainder, to which other NFs approached closely, were labeled mostly with NE14, SMI31, or N52. Thus, when NFs appear singly, NF-H in their projections or cross-bridges with other organelles is not phosphorylated, while when NFs are bundled, NF-H is phosphorylated in crossbridges between NF core filaments. These data may explain why the NF-H is heavily phosphorylated in axons, where NFs are abundant, and not in dendrites and perikarya, where NFs are sparse. Wiley-Liss, Inc.     

Differential titin isoform expression in human skeletal muscle

Mammalian skeletal muscle expresses at least two isoforms of the cytoskeletal protein titin (connectin; MW ≈ 3000 kDa). These isoforms are associated with different passive force curves, and thus may affect physical performance. To study the distribution of titin and its possible influence on performance in humans, muscle biopsies were obtained from 15 males (X ± SE; age = 25.4 ± 2.9 years, height = 177.7 ± 1.8 cm, weight = 76.5 ± 2.2 kg). Two biopsies were obtained on separate occasions from both the right and left vastus lateralis, and one biopsy each from the lateral head of the right gastrocnemius and the right soleus, with all biopsies handled identically. Fibre type analyses were performed via mATPase histochemistry. Expression of titin and myosin heavy chain isoforms were determined by SDS-PAGE. Titin bands in the resulting gels were highly repeatable and were verified by migration patterns, as well as Western blot analysis. Two groups of subjects were identified: group 1 (n=10) expressed only one titin isoform (titin-1) in all biopsies, and group 2 (n=5) expressed two titin isoforms (titin-1 and titin-2) in all biopsies. No significant differences (P> 0.05) between groups were observed for percentage fibre types, percentage fibre type areas, fibre type cross-sectional areas, and percentage myosin heavy chain expression when comparing individual muscles, sampling times or bilateral comparisons. This is the first report of differential titin isoform expression in healthy, mature human skeletal muscle, but it is not clear why this occurs or what influence this may have on performance.     

Relationship of actin filament assembly to clearance of fibrinogen gold, GPIIb-IIIa complexes on spread platelets

Abstract. The present study has evaluated the influence of high concentrations of cytochalasins B and E on the detergent-resistant actin levels in fully spread platelets by PAGE gel electrophoresis, and the effects of the two inhibitors of new actin filament assembly on translocation of fibrinogen gold (Fgn/Au) labelled GPIIb-IIIa receptors on the surface-activated cells. Concentrations of 10^{- 4}m and 10^-5 m cytochalasin B and E reduced detergent-resistant actin in fully spread platelets to levels present in resting discoid platelets in suspension. Despite reduction of actin filaments to levels in resting cells, cytochalasin B did not prevent translocation of Fgn/Au from platelet margins into channels of the open canalicular system (OCS). Similar concentrations of cytochalasin E completely blocked translocation of receptor-ligand complexes and produced a patching phenomenon not observed in previous studies. Rinsing of the spread cells to remove cytochalasin, followed by incubation of the treated platelets in Hank's buffered salt solution (HBSS) restored levels of detergent-resistant actin to those found in untreated, spread platelets. Resting grids of 10 ⁵ m cytochalasin E-treated platelets on drops of HBSS for 15min restored their ability to clear FGN/Au linked to GPIIb-IIIa from exposed surfaces to the OCS, but 10^-4 m cytochalasin E-treated cells remained anergic after incubation on drops of HBSS. Thus a fully assembled cytoplasmic actin filament cytoskeleton does not appear to be essential for translocating receptor-ligand complexes on the platelet surface to the OCS, nor does its presence guarantee that the ability to clear GPIIb-IIIa receptors will be restored.