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Background: Proteinuria, one of the main manifestations of nephrotic syndrome, is an important risk factor for the progression of renal diseases. Podocyte foot processes (FPs) injury induces proteinuria in most renal diseases. The podocyte cytoskeleton plays important roles in maintaining the normal morphology of FPs. However, the underlying cytoskeletal component that initiates and regulates the dynamic changes of FPs is still unclear. Here, the involved podocyte cytoskeletal molecules were explored on different days in puromycin aminonucleoside nephropathy rats., Methods: Microarray analysis of isolated glomeruli was performed at Day 2, Day 10 and Day 15 in puromycin aminonucleoside nephropathy rats. Cytoskeletal genebank was established by sorting with the keyword 'cytoskeleton' from PUBMED genebank to identify the differential cytoskeleton genes. Microarray results were further confirmed by real-time PCR, western blot and double immunolabelling to validate their localizations., Results: Nine different cytoskeletal genes were found to be involved in the dynamic changes of FPs in puromycin aminonucleoside nephropathy rats, including six up-regulated (Tagln, Actr2, Dnm3, Arc, Vcl and Birc5) and three down-regulated (Krt2-7, Nebl and Tnnc1). The differential expression of transgelin, survivin, arp2, cytokeratin7 and vinculin was verified by real-time PCR and western blot. Double immunolabelling revealed that five cytoskeletal proteins indeed colocalized with podocyte specific markers synaptopodin or alpha-actinin-4. In addition, similar expression and distribution changes were detected in patients with proteinuric renal diseases and puromycin aminonucleoside-treated podocytes., Conclusions: We identified five novel podocyte cytoskeletal proteins and found that they were associated with the dynamic changes of FPs in podocyte injury.

Objective: Dystrophin is an integral membrane protein that stabilizes the sarcolemmal membrane integrity, and its loss may be involved in the mechanism of ischemia and reperfusion injury. It has been reported that ischemic preconditioning is related to the preservation of membrane dystrophin during ischemia and reperfusion. Preconditioning with nicorandil, a mitochondrial K(ATP) channel opener, may attenuate the injury by preventing a disturbance in the level of this membrane-associated protein., Methods: The isolated rat hearts were subjected to 60 min of cardioplegic arrest, followed by 60 min of reperfusion. The hearts were divided into the following three groups according to the drugs given before cardioplegic arrest. The control group received saline intravenously 30 min before heart isolation. The nicorandil group received nicorandil (0.3 mg/kg) intravenously 30 min before isolation. The 5-HD group received 5-hydroxydecanoate (1 mg/kg) intravenously, a mitochondrial K(ATP) channel blocker, 5 min before nicorandil administration. Cardiac function, myocardial metabolism, dystrophin distribution and protein levels of dystrophin were assessed before and after cardioplegic arrest., Results: The nicorandil group showed significantly better cardiac function and a significant reduction in creatine kinase release during reperfusion. After 60 min of cardioplegic arrest, dystrophin, which was distributed predominantly in the sarcolemmal membrane before ischemia, was translocated to the costameric cytoskeleton in all groups. During reperfusion, the level of membrane dystrophin remained decreased in the majority of cardiomyocytes in the control and 5-HD groups, whereas it was restored to nearly the baseline level in the nicorandil group. The immunoblot analysis supported this result., Conclusions: Depletion of sarcolemmal membrane dystrophin occurred during cardioplegic arrest and reperfusion. Nicorandil preconditioning may attenuate ischemia and reperfusion injury by maintaining the membrane structural integrity.

The seminiferous epithelium contains unique actin related cell-cell junctions, termed ectoplasmic specializations (ESs). Turnover of these junctions is fundamental to sperm release and to movement of spermatocytes from basal to adluminal compartments of the epithelium during spermatogenesis. In this study we report several novel observations related to the spatial and temporal distribution of integrin-related signaling molecules at ESs. We confirm the presence of beta(1)-integrin at these sites and further demonstrate co-localization of integrin linked kinase (ILK). beta(1)-Integrin and ILK were shown by immunoprecipitation to associate in whole cell lysates of seminiferous epithelium. This observation provides the first evidence for a direct beta(1)-integrin/ILK interaction in noncultured epithelium. Pan-cadherin and beta-catenin antibodies did not react at ESs. Rather, antibodies reacted with desmosome-like junctions that are present both at basal junctional complexes between Sertoli cells and at sites of attachment to spermatogenic cells. Focal adhesion kinase (FAK), a known integrin-associated molecule, did not codistribute with beta(1)-integrins and did not associate with these adhesion molecules in immunoprecipitation studies. Although FAK was expressed in the epithelium, it appeared to be limited to the cytoplasm of early spermatogenic cells. Significantly, polyclonal antibodies against phosphotyrosine-containing residues reacted strongly at ESs, with highest levels detected during sperm release and turnover of basal junction complexes. Our observations indicate that ESs share cell signaling features both of cell-cell junctions and of cell-extracellular matrix junctions.

A 260-kDa protein, termed cgABP260, which localized in the dense plaques and dense bodies of smooth muscle cells, was found in a low-salt alkaline extract of chicken gizzard smooth muscle. An antibody against cgABP260 was used to screen a chicken gizzard cDNA library, and the nucleotide sequence of the partial cDNA encoding this protein was determined. Comparison of predicted amino acid sequences revealed that the protein had significant homology with human ABP-280 and chicken retina filamin [Barry, C.P. et al. (1993) J. Biol. Chem. 268, 25577-25586], but despite the high homology, cgABP260 was immunologically distinguishable from filamin. Immunoblot analysis showed that an anti-cgABP260 antibody reacted exclusively with the cgABP260 band of smooth, skeletal, and cardiac muscle tissues. By indirect immunofluorescence, the membrane-affinity-purified antibody against cgABP260 intensely stained the dense plaques of the isolated smooth muscle cells. Immunoelectron microscopy showed that immunogold particles representing cgABP260 were found abundantly on the dense plaques and less abundantly on the dense bodies. Its amino acid sequence, molecular size, immunological reactivity, and localization in smooth muscle thus indicated that cgABP260 is a new component of the dense plaques and dense bodies of smooth muscle cells.

The follicular histoarchitecture of the thyroid forms the anatomical basis for thyroid physiology and is commonly disturbed in diseases of the thyroid. We have used cultured porcine thyroid cells to study thyroid epithelial morphogenesis and its regulation. When cultured in the presence of TSH, freshly isolated thyroid cells reorganize to form follicles within three-dimensional cell aggregates. However, when established follicles are washed into TSH-free medium, thyroid cells spread and migrate to convert follicles into confluent epithelioid monolayers, activating morphogenetic mechanisms, such as cell locomotility, that may be relevant to thyroid inflammation and tumor invasiveness. The phenomenon of follicle to monolayer conversion, therefore, provides an opportunity to identify morphogenetic mechanisms that 1) must be tonically inhibited to maintain follicular organization and 2) may contribute to pathogenetic disturbances of follicular architecture when functioning aberrantly. In this study we found that follicle to monolayer conversion is associated with an increase in cellular phosphotyrosine. This was particularly evident at nascent focal adhesions (cell-substrate adhesive junctions) and later at cell-cell junctions. Focal adhesion assembly was accompanied by reorganization of the actin cytoskeleton, with the appearance of prominent stress fibers. Genistein, a potent inhibitor of protein tyrosine kinases, inhibited the accumulation of phosphotyrosine, focal adhesion assembly, and follicle to monolayer conversion. We conclude that tyrosine phosphorylation exerts an important influence on thyroid epithelial organization in culture, at least partly mediated through regulation of focal adhesion assembly.

Specialized junctional binding of step 8 spermatids to Sertoli cells is an important spermiogenic event. In the hypophysectomized (Hypox) rat, the daily sperm production (DSP) is reduced and Sertoli cells become binding incompetent. Delayed replacement of testosterone (DT-Hypox) does not restore the normal DSP and Sertoli cells remain binding incompetent. In this study, DT-Hypox rats received FSH daily for 2 days to 3 wk concurrent with delayed testosterone replacement and were killed 8 wk after the initiation of hormone treatment. The DT-Hypox rat treated with FSH for 2 days to 2 wk had a significantly reduced DSP. Sertoli cells remained binding incompetent as evidenced by structurally abnormal ectoplasmic specializations and an abnormal pattern of f-actin and vinculin immunostaining. The DT-Hypox rat treated with FSH for 3 wk had a normal DSP. Sertoli cells were binding-competent as evidenced by structurally intact ectoplasmic specializations and the normal pattern of f-actin and vinculin immunostaining. The results indicate that the "priming" effect of FSH necessary to restore normal spermiogenesis is associated with restoration of the junction-related Sertoli cell cytoskeleton (i.e., FSH induction of binding competency) expressed as intact ectoplasmic specializations and peripheral distribution of f-actin and vinculin.

This report examines the relationship between aromatase activity and progesterone production and the expression of actin and vinculin in rat granulosa cells, exposed to insulin, follicle stimulating hormone (FSH) and human chorionic gonadotrophin (HCG). Granulosa cells of pre-antral follicles from juvenile rats treated with diethylstilbestrol (DES) were cultured on collagen A-coated plastic coverslips in serum-free medium. At a moderate or low level of steroidogenesis (FSH alone), the expression of vinculin was diminished while vinculin plaques disappeared completely. At a high level of steroidogenesis (both FSH and insulin), actin in stress fibre form was also decreased considerably. Under conditions of progesterone production (pretreatment with FSH and subsequent incubation with HCG), a concomitant increase of actin in soluble form was found. It is concluded from these studies that the higher the steroidogenesis level of the granulosa cells, the lower the organization of the microfilaments vinculin and actin, which are regulated independently of each other.

Resorption and migration phases alternate in the life of the osteoclast. We have previously described a specific microfilament structure at the attachment sites in resorbing osteoclasts. In the present study we have examined microfilaments and microtubules in both resorbing and migrating rat osteoclasts cultured on bone slices. In migrating osteoclasts microfilaments form so-called podosome structures containing vinculin, talin, and F-actin at the paramarginal area of the cell. When the osteoclast prepares itself for resorption, the podosomes gather to a certain area and form a broad ring around the area, which is then resorbed. In the resorbing osteoclast, vinculin and talin form a continuous double circle, which may be partially formed by podosomes, and between these double circles a broad zone is formed by F-actin. Narrow vinculin and F-actin rings were found in osteoclasts at the end of the resorption phase. The different configurations of microfilaments in 1 and 2 day cultures were correlated in terms of their relationship to the resorption lacunae. The vitamin A derivative isotretinoin significantly stimulated resorption and increased the number of microfilament configurations associated with the resorption pits. On the other hand, Bt2cAMP abolished resorption and prevented the formation of a specific ring structure of microfilaments. Based on these data, a kinetic model of the whole migration-resorption cycle of the osteoclast cultured on the bone slice is presented. With alpha-tubulin stainings of microtubules two different cytoskeletal organizations were observed. In migrating osteoclasts, microtubules were evenly distributed over the whole cell. In the resorbing osteoclast, there was a noticeable concentration of these cytoskeletal structures at cytoplasmic sites closest to the resorption lacuna. This orientation of microtubules may reflect the active secretory function of the resorbing osteoclast.