Directing the axis of cell division toward extrinsic and intrinsic cues plays a fundamental role in morphogenesis, asymmetric cell division, and stem cell self-renewal. Recent studies highlight the misorientation of the cell division axis as a cause of mammalian diseases, including polycystic kidney disease. Although the core regulators for oriented cell division have been identified in invertebrate model systems, we still have an imprecise picture of the relevant signaling networks in the mammalian system. The reasons for this include the lack of established approaches in mammalian cells to survey the molecules required for the spindle orientation. Here we summarize our recent study on a genome-scale RNA-mediated interference screen of human kinases to identify a new player for the oriented cell division in both culture cells and developing mammalian tissues.

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The blood-nerve barrier (BNB) is a highly specialized unit that maintains the microenvironments of the peripheral nervous system. Since the breakdown of the BNB has been considered a key step in autoimmune neuropathies such as Guillain-Barré syndrome and chronic inflammatory demyelinating polyraduculoneuropathy, it is important to understand the cellular properties of the peripheral nerve microvascular endothelial cells (PnMECs) which constitute the BNB. For this purpose, we established an immortalized cell line derived from human PnMECs. The human PnMECs were transduced with retroviral vectors encoding the temperature-sensitive SV40 large T antigen and human telomerase. This cell line, termed FH-BNB, showed a spindle fiber-shaped morphology, expression of von Willebrand factor and uptake of acetylated low density lipoprotein. These cells expressed tight junction proteins including occludin, claudin-5, ZO-1 and ZO-2 at the cell-cell boundaries. P-glycoprotein and GLUT-1 were also detected by a Western blot analysis and the cells exhibited the functional expression of p-glycoprotein. In addition, transendothelial electrical resistance experiments and paracellular permeabilities of sodium fluorescein and fluorescein isothiocyanate-labeled dextran of molecular weight 4 kDa across these cells demonstrated that FH-BNBs had functional tight junctions. These results indicated that FH-BNBs had highly specialized barrier properties and they might therefore be a useful tool to analyze the pathophysiology of various neuropathies.

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Recent studies have revealed various biological functions for repetitive sequences, which make up about half of the human genome. One such sequence, major satellites, which are tandem repetitive sequences adjacent to the centromere, have been shown to be a kinetochore component that plays a role in the formation and function of the pericentric heterochromatin necessary for mitosis. However, it is unknown whether these regions also play a role in post-mitotic cells. Here, we show that, during neuronal differentiation, the heterochromatin domains that include major satellite regions become both enriched with the active histone modification lysine-4 trimethylation of histone H3, and more sensitive to nuclease, both of which suggest increased activation of this area. Further supporting this notion, we also found that transcription from major satellite regions is significantly increased during neuronal differentiation both in vitro and in vivo. These results together suggest that the structural and transcriptional state of major satellite regions changes dramatically during neuronal differentiation, implying that this region might play a role in differentiating neurons.

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Intracellular vesicle traffic plays an essential role in the establishment and maintenance of organelle identity and biosynthetic transport. We have identified α-taxilin as a binding partner of the syntaxin family, which is involved in intracellular vesicle traffic. Recently, we have found that α-taxilin is over-expressed in malignant tissues including hepatocellular carcinoma and renal cell carcinoma. However, a precise role of α-taxilin in intracellular vesicle traffic and carcinogenesis remains unclear. Then, we first investigated here the intracellular distribution of α-taxilin in Hela cells. Immunofluorescence studies showed that α-taxilin distributes throughout the cytoplasm and exhibits a tubulo-vesicular pattern. Biochemical studies showed that α-taxilin is abundantly localized on intracellular components as a peripheral membrane protein. Moreover, we found that α-taxilin distributes in microtubule-dependent and syntaxin-independent manners, that α-taxilin directly binds to polymerized tubulin in vitro, and that N-ethylmaleimide but not brefeldin A affects the intracellular distribution of α-taxilin. These results indicate that α-taxilin is localized on intracellular components in a syntaxin-independent manner and that the α-taxilin-containing intracellular components are associated with the microtubule cytoskeleton and suggest that α-taxilin functions as a linker protein between the α-taxilin-containing intracellular components and the microtubule cytoskeleton.

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The present study focuses on mechanotransduction in mesenchymal stem cells (MSCs) in response to matrix elasticity. By using photocurable gelatinous gels with tunable stiffness, proteomic profiles of MSCs cultured on tissue culture plastic, soft (3 kPa) and stiff (52 kPa) matrices were deciphered using 2-dimensional differential in-gel analysis (2D-DIGE). The DIGE data, tied to immunofluorescence, indicated abundance and organization changes in the cytoskeletonal proteins as well as differential regulation of important signaling-related proteins, stress-responsing proteins and also proteins involved in collagen synthesis. The major CSK proteins including actin, tubulin and vimentin of the cells cultured on the gels were remarkably changed their expressions. Significant down-regulation of α-tubulin and β-actin can be observed on gel samples in comparison to the rigid tissue culture plates. The expression abundance of vimentin appeared to be highest in the MSCs cultured on hard gels. These results suggested that the substrate stiffness significantly affects expression balances in cytoskeletal proteins of MSCs with some implications to cellular tensegrity.

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Small GTPases ARF1 and ARF3 localize mainly to the Golgi apparatus, where they trigger formation of coated carrier vesicles. We previously showed that BIG2, a guanine nucleotide exchange factor specific for ARF1 and ARF3, localizes not only to the trans-Golgi network (TGN) but also to recycling endosomes, where it is involved in regulating the integrity of recycling endosomes. However, it is not yet clear whether ARF1 and ARF3 act downstream of BIG2 to ensure endosome integrity. In this study, we show that EGFP-tagged ARF1 and ARF3 localize to endosomal compartments containing endocytosed transferrin. We further demonstrate that simultaneous depletion of ARF1 and ARF3 induces tubulation of recycling endosomal compartments positive for transferrin receptor, Rab4, and Rab11, but does not significantly affect the integrity of the Golgi apparatus or early or late endosomes. Moreover, the simultaneous depletion of ARF1 and ARF3 suppresses recycling of transferrin but does not affect either its endocytosis or the retrograde transport of TGN38 from early/recycling endosomes to the TGN. In addition, depletion of ARF1 and ARF3 does not affect retrograde transport of CD4-furin from late endosomes to the TGN, or of endocytosed EGF from late endosomes to lysosomes. These results indicate that ARF1 and ARF3 are redundantly required for the integrity of recycling endosomes, and that they regulate transferrin recycling from endosomes to the plasma membrane, but not retrograde transport from endosomal compartments to the TGN.

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The small GTP-binding protein Rho plays a crucial role in a wide variety of cellular functions through various effector proteins. Rho-kinase is a key effector protein of Rho, which is composed of two isoforms, ROCK1 and ROCK2. To clarify the site of action of ROCK1 and ROCK2, we performed immunofluorescence and immunoelectron microscopic analyses using isoform-specific antibodies in mouse tissues. In the large and small intestines, ROCK1 immunoreactivity was predominantly identified in epithelial cells, and ROCK2 immunoreactivity was negligible. In these epithelial cells, ROCK1 immunoreactivity was distributed on plasma membranes, while ROCK1 immunogold signals were localized at cell-cell contacts and cell adhesion sites, especially at the adherens junctions at the ultrastructural level. In the bladder epithelium, however, ROCK1 and ROCK2 signals were identified at intermediate filaments, and ROCK2 signals were also observed in nuclei. In the three types of muscular cells—smooth, cardiac, and skeletal muscle cells—ROCK1 and ROCK2 also showed differential distribution. ROCK1 signals were localized at actin filaments, plasma membranes, and vesicles near plasma membranes in smooth muscle cells; at the lysosomes in skeletal muscle cells; and were undetectable in cardiac muscle cells. ROCK2 signals were localized at actin filaments and centrosomes in smooth muscle cells, at intercalated discs in cardiac muscle cells, and at Z-discs and sarcoplasmic reticulum in skeletal muscle cells. In the brain, ROCK1 immunoreactivity was distributed in glia, whereas ROCK2 immunoreactivity was observed in neurons. These results indicate that the two isoforms of Rho-kinase distribute differentially to accomplish their specific functions.

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Misfolded proteins in the endoplasmic reticulum (ER) are dislocated out of the ER to the cytosol, polyubiquitinated, and degraded by the ubiquitin-proteasome system in a process collectively termed ER-associated degradation (ERAD). Recent studies have established that a mammalian ER-localized transmembrane J-protein, DNAJB12, cooperates with Hsc70, a cytosolic Hsp70 family member, to promote the ERAD of misfolded membrane proteins. Interestingly, mammalian genomes have another J-protein called DNAJB14 that shows a high sequence similarity to DNAJB12. Yet, very little was known about this protein. Here, we report the characterization of DNAJB14. Immunofluorescence study and protease protection assay showed that, like DNAJB12, DNAJB14 is an ER-localized, single membrane-spanning J-protein with its J-domain facing the cytosol. We used co-immunoprecipitation assay to find that DNAJB14 can also specifically bind Hsc70 via its J-domain to recruit this chaperone to ER membrane. Remarkably, the overexpression of DNAJB14 accelerated the degradation of misfolded membrane proteins including a mutant of cystic fibrosis transmembrane conductance regulator (CFTRΔF508), but not that of a misfolded luminal protein. Furthermore, the DNAJB14-dependent degradation of CFTRΔF508 was compromised by MG132, a proteasome inhibitor, indicating that DNAJB14 can enhance the degradation of a misfolded membrane protein using the ubiquitin-proteasome system. Thus, the mammalian ER possesses two analogous J-proteins (DNAJB14 and DNAJB12) that both can promote the ERAD of misfolded transmembrane proteins. Compared with DNAJB12 mRNA that was widely expressed in mouse tissues, DNAJB14 mRNA was expressed more weakly, being most abundant in testis, implying its specific role in this tissue.

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