Regulation of blood–testis barrier by actin binding proteins and protein kinases

The blood–testis barrier (BTB) is an important ultrastructure in the testis, since the onset of meiosis and spermiogenesis coincides with the establishment of a functional barrier in rodents and humans. It is also noted that a delay in the assembly of a functional BTB following treatment of neonatal rats with drugs such as diethylstilbestrol or adjudin also delays the first wave of spermiation. While the BTB is one of the tightest blood–tissue barriers, it undergoes extensive remodeling, in particular, at stage VIII of the epithelial cycle to facilitate the transport of preleptotene spermatocytes connected in clones across the immunological barrier. Without this timely transport of preleptotene spermatocytes derived from type B spermatogonia, meiosis will be arrested, causing aspermatogenesis. Yet the biology and regulation of the BTB remains largely unexplored since the morphological studies in the 1970s. Recent studies, however, have shed new light on the biology of the BTB. Herein, we critically evaluate some of these findings, illustrating that the Sertoli cell BTB is regulated by actin-binding proteins (ABPs), likely supported by non-receptor protein kinases, to modulate the organization of actin microfilament bundles at the site. Furthermore, microtubule-based cytoskeleton is also working in concert with the actin-based cytoskeleton to confer BTB dynamics. This timely review provides an update on the unique biology and regulation of the BTB based on the latest findings in the field, focusing on the role of ABPs and non-receptor protein kinases.

EXPRESSION AND LOCATION OF VASP IN HUMAN ENDOTHELIAL CELLS: EFFECT OF A LAMINAR SHEAR STRESS

Recent elucidation of the primary VASP (vasodilator-simulated phosphoprotein) and identity of VASP binding proteins suggests that VASP is an important component of focal contacts which links signal transduction pathways and elements controlling cell motility. The aim of our study was to evaluate shear-stress-induced changes of VASP expression and localization in ECs. We showed, by western blotting measurements, that the shear stress involve some modifications on the expression of phosphorilated (50 kD) and non phosphorilated (46kD) VASP. Moreover, a fluorescence double-labeling shows the location of VASP on the actin fibers. At rest, ECs showed an array of microfilament bundles of the actin fibers and VASP were along their entire length. After exposure to shear stress, the stress fibers appeared and were oriented along with the flow. There were a thicker expression of VASP than in control, targeted to the ends of stress fibres. This seems to be an adjustment of the cells towards the mechanical stresses. These results suggest that VASP is a potential important component which participates in the regulation of cell actin remodelling induced by shear flow. VASP were involved in the mechano-transduction pathways.

Changes in actin microfilament arrays in developing pea root nodule cells

Various microorganisms that form symbiotic associations with plant roots alter the cytoskeleton of host cells. The objective of this study was to determine the organization of actin microfilaments in developing Pisum sativum L. (pea) root nodule cells at various stages after infection by Rhizobium leguminosarum bv. viciae. Fluorescently labelled microfilaments in uninfected pea root nodule cells occur in association with the nucleus, along cytoplasmic strands, and as long microfilament bundles randomly organized in the cortex of the cell. These actin arrays are also present in recently infected cells that have been invaded by an infection thread and contain a small number of bacteroids. In addition, the recently infected cells contain diffuse cytoplasmic actin, long actin microfilament bundles near the vacuole, and a nuclear-associated network of microfilament bundles. In older infected cells, the predominant array is a network of cytoplasmic microfilaments that are wavy and extend in multiple directions within the cell; the network is equally abundant in all regions of the cytoplasm and may interact with the bacteroids and organelles. Thus, actin microfilaments reorganize during the pea root nodule infection process to form distinct arrays whose organization depends on the stage of infection.Key words: nodule, actin microfilaments, Rhizobium, pea, symbiosis.

Immunoexpression of Villin in Neuroendocrine Tumors and Its Diagnostic Implications

Background.—Villin, a 95-kd cytoskeletal protein associated with axial microfilament bundles of brush border microvilli, is mostly restricted to intestinal glandular tumors. Villin immunoexpression was recently observed in a small number of carcinoids of the intestinal tract and lung, but its significance in a broad category of neuroendocrine tumors has not been evaluated. Design.—A total of 114 neuroendocrine tumors of different origins were tested for villin expression. They included gastrointestinal carcinoids (n = 30), lung carcinoids (n = 15), small cell carcinomas of the lung (n = 24), small cell carcinomas of other sites (n = 15), islet cell tumors (n = 8), Merkel cell carcinomas (n = 6), paragangliomas (n = 6), and others (n = 10). Nine round cell sarcomas were tested as well. Results.—Villin immunoreactivity was present in 85% of gastrointestinal carcinoids and small cell carcinomas, but was found in only 40% of lung carcinoids. Other tumors tested were virtually negative for villin. In general, while cytoplasmic reactivity was most common, a characteristic apical membranous pattern simulating brush border was seen in 76% of the gastrointestinal carcinoids and in 50% of the lung carcinoids. Conclusions.—We found that villin was predominantly restricted to gastrointestinal neuroendocrine tumors (excluding islet cell tumors), although a small number of bronchial carcinoids may be positive as well. These results suggest a role for villin in the differential diagnosis of neuroendocrine tumors.

Conformational difference between nuclear and cytoplasmic actin as detected by a monoclonal antibody

Using a reconstituted complex of profilin and skeletal muscle actin as an antigen, we generated a monoclonal mouse antibody against actin, termed 2G2. As revealed by immunoblots of proteolytic actin fragments and by pepscan analysis, the antibody recognises a nonsequential epitope on actin which is located within three different regions of the sequence, consisting of aa131-139, aa155-169, and aa176-187. In the actin model derived from X-ray diffraction, these sequences lie spatially close together in the region of the nucleotide-binding cleft, but do not form a coherent patch. In immunoblots, 2G2 reacts with all SDS-denatured actin isoforms and with actins of many vertebrates. In contrast, its immunofluorescence reactivity is highly selective and fixation-dependent. In fibroblasts and myogenic cells, fixed and extracted by formaldehyde/detergent, stress fibres or myofibrils, respectively, remained unstained. Likewise, after microinjection into living cells, 2G2 did not bind to such microfilament bundles. Extraction of myosin and tropomyosin did not alter this pattern indicating that the lack in reactivity is probably not due to epitope-masking by actin-binding proteins. More likely, the reason for the lack of reactivity with filamentous actin is that its epitope is not accessible in F-actin. However, the antibody revealed a distinct pattern of nuclear dots in differentiated myogenic cells but not in myoblasts, and of fibrillar structures in nuclei of Xenopus oocytes. In contrast, after methanol treatment, a 2G2-specific staining of stress fibres and myofibrils was observed, but no nuclear dot staining. We conclude that 2G2, in addition to binding to SDS- and methanol-denatured actin, recognises a specific conformation of native actin which is present in the nucleus and specified by compaction of the antibody-reactive region into a coherent patch. This conformation is apparently present in differentiated myogenic cells and oocytes, but not in cytoplasmic actin filament bundles.

Specific Localization of Serine 19 Phosphorylated Myosin II during Cell Locomotion and Mitosis of Cultured Cells

Phosphorylation of the regulatory light chain of myosin II (RMLC) at Serine 19 by a specific enzyme, MLC kinase, is believed to control the contractility of actomyosin in smooth muscle and vertebrate nonmuscle cells. To examine how such phosphorylation is regulated in space and time within cells during coordinated cell movements, including cell locomotion and cell division, we generated a phosphorylation-specific antibody. Motile fibroblasts with a polarized cell shape exhibit a bimodal distribution of phosphorylated myosin along the direction of cell movement. The level of myosin phosphorylation is high in an anterior region near membrane ruffles, as well as in a posterior region containing the nucleus, suggesting that the contractility of both ends is involved in cell locomotion. Phosphorylated myosin is also concentrated in cortical microfilament bundles, indicating that cortical filaments are under tension. The enrichment of phosphorylated myosin in the moving edge is shared with an epithelial cell sheet; peripheral microfilament bundles at the leading edge contain a higher level of phosphorylated myosin. On the other hand, the phosphorylation level of circumferential microfilament bundles in cell–cell contacts is low. These observations suggest that peripheral microfilaments at the edge are involved in force production to drive the cell margin forward while microfilaments in cell–cell contacts play a structural role. During cell division, both fibroblastic and epithelial cells exhibit an increased level of myosin phosphorylation upon cytokinesis, which is consistent with our previous biochemical study (Yamakita, Y., S. Yamashiro, and F. Matsumura. 1994. J. Cell Biol. 124:129–137). In the case of the NRK epithelial cells, phosphorylated myosin first appears in the midzones of the separating chromosomes during late anaphase, but apparently before the formation of cleavage furrows, suggesting that phosphorylation of RMLC is an initial signal for cytokinesis.

Plant and animal profilins are functionally equivalent and stabilize microfilaments in living animal cells

We have analyzed the degree of functional similarity between birth and mammalian profilins, two members of the profilin family which show only a moderate sequence homology (22%) in living animal cells. The plant profilin, derived from birch pollen, was stably expressed in BHK-21 cells. Plant and endogenous profilin synthesis and cellular distribution were monitored by specific monoclonal antibodies. Quantitation of profilin and actin on calibrated immunoblots showed that two stable clones contained in total 1.4 and 2.0 times as much profilin as the parental cells. Using double fluorescence and confocal laser scanning microscopy, it was seen that the endogenous and the plant profilin colocalized with dynamic microfilaments, in particular with F-actin-rich foci and cortical microfilament webs of spreading cells, with dynamic microfilament bundles induced by serum deprival, and with cytochalasin D- and latrunculin-induced transient F-actin aggregates. The increase in the overall profilin concentration correlated with a significantly higher resistance of actin filaments to these drugs. Our data indicate that even profilins of highly distant evolutionary origin can functionally substitute for each other and support the hypothesis that in animal cells, profilins are engaged in regulating either the stability or the kinetic properties of actin filaments.