Journal of cell science. Supplement最新文献

MDCK cells maintain the polarized distribution of surface proteins mainly by sorting the newly synthesized proteins in the trans-Golgi network (TGN). In order to identify the components of the putative sorting machinery and to study factors that affect the sorting process, we have developed an in vitro system that reconstitutes the transport of viral glycoproteins from the TGN to the apical or basolateral surface. We have used this system to study effects of membrane impermeable reagents (such as peptides and antibodies) on the polarized transport. We observed that reagents affecting the stimulatory class (Gs) of heterotrimeric GTP binding proteins (G proteins) influenced the apical but not the basolateral transport. In contrast, reagents specific for the inhibitory class of G proteins (Gi) affected the basolateral but not the apical transport. These results show that the heterotrimeric G proteins differentially regulate the two pathways of polarized transport. The G proteins may regulate the process of polarized sorting of proteins in a fashion analogous to their role in signal transduction by providing a communication link with the cytosolic side of the membrane.

It has been realized for some time that the loss of epithelial differentiation in carcinomas, which is accompanied by higher mobility and invasiveness of the tumor cells, is a consequence of reduced intercellular adhesion. A variety of recent reports have indicated that the primary cause for the 'scattering' of the cells in invasive carcinomas is a loss of the integrity of intercellular junctions. Thus, defects in expression or structure of several components of the epithelial adherens junctions (e.g. E-cadherin, alpha-catenin) can occur, and our increased knowledge about the molecules of the junctions allows an explanation of these defects in molecular terms in some of the cases. Furthermore, tyrosine phosphorylation of junctional components (e.g. beta-catenin) appears to play a role in the assembly and disassembly of cell-cell contacts. Some of the effectors of epithelial junction formation are tyrosine protein kinases, e.g. the scatter factor/hepatocyte growth factor receptor c-Met, the FGF receptors and the pp60src kinase. The importance of tyrosine phosphorylation in junctions during tumor development is becoming increasingly evident.

Nerve growth factor (NGF) represents a family of structurally related trophic factors, including brain-derived neurotrophin factor (BDNF), neurotrophin-3 (NT-3), NT-4, and NT-5. These neurotrophin factors interact with two classes of receptors, the trk receptor tyrosine kinase family, and the low affinity p75 neurotrophin receptor. To study potential ligand-receptor interactions, recombinant trk fusion proteins have been constructed, and pan-trk polyclonal antisera directed against the cytoplasmic tyrosine kinase domain have been generated. The recombinant proteins were assessed for in vitro kinase activity and for the ability of K-252a to inhibit phosphorylation. Antibodies made against the fusion protein recognize all trk family members, and are effective in immunoprecipitation of affinity-crosslinked receptors. Comparative crosslinking indicates that NGF can recognize all trk receptor members, illustrating the large number of potential ligand-receptor interactions between neurotrophins and their receptors.

Genetic analysis in Drosophila has led to the identification of several proteins that mediate cell-cell interactions controlling the fate and proliferation of epithelial cells. These proteins are localized or enriched in the adherens and septate junctions at the apical end of the lateral membranes between cells. The proteins localized or enriched at adherens junctions include Notch, which is important for the cell interactions controlling neuroblast and bristle patterning; Boss and sevenless, which are required for the cell interaction that establishes the R7 photoreceptor cell; and Armadillo, required for the wingless-dependent cell interactions that control segment polarity and imaginal disc patterning. Proteins localized at septate junctions include the product of the tumor suppressor gene dlg, which is required for septate junction formation, apical basal cell polarity, and the cell interactions that control proliferation. The results suggest that the cell signalling events important for cell fate determination and for cell proliferation control in epithelia occur at the apical junctions. The migration of the nucleus to the apical surface of the epithelium for mitosis may enable it to interact directly with the junction-associated signalling mechanisms.

Neurons have at least two pathways of regulated secretion, which involve two classes of secretory organelles: typical synaptic vesicles (SVs) and large dense-core vesicles. Large dense-core vesicles store and secrete peptide neurotransmitters and amines, and may be seen as the neuronal counterpart of secretory granules of endocrine cells. SVs are highly specialized secretory organelles, which store and secrete non-peptide hormones and play a dominant role in the fast, point-to-point signalling typical of the nervous system. Microvesicles that share a variety of biochemical and functional similarities with SVs (synaptic-like microvesicles) have recently been described in endocrine cells. SVs and synaptic-like microvesicles are closely related to vesicular carriers of the receptor-mediated recycling pathway. They undergo repeated cycles of exo-endocytosis, which are thought to involve endosomal intermediates. In mature neurons, SVs are concentrated in axon endings. To gain insight into the mechanisms responsible for SV targeting, we have studied the traffic of SV proteins in both endocrine cells and developing hippocampal neurons in primary culture at different stages of differentiation. Additionally, the distribution of the SV protein synaptophysin, when expressed by transfection in fibroblastic cells or in polarized epithelial cells (MDCK cells), was investigated. SV proteins are already present in developing neurons at stages preceding the establishment of neuronal polarity. As axons and dendrites form, SV proteins are found in both types of processes, although they become progressively more concentrated in the axon. Throughout these developmental stages SVs undergo active exo-endocytotic recycling.(ABSTRACT TRUNCATED AT 250 WORDS)

Motor neuron disease is clinically characterized by progressive muscle wasting leading to total muscle paralysis. A long history of pathological study of patients has firmly established that the primary lesion site is in spinal and cortical motor neurons. In addition to the wide-spread loss of these neurons, neuronal abnormalities including massive accumulation of neurofilaments in cell bodies and proximal axons have been also widely observed, particularly in the early stages of the disease. To test whether high accumulation of neurofilaments directly contributes to the pathogenic process, transgenic mice that produce high levels of neurofilaments in motor neurons have been generated. These transgenic mice show most of the hallmarks observed in motor neuron disease, including swollen perikarya with eccentrically localized nuclei, proximal axonal swellings, axonal degeneration and severe skeletal muscle atrophy. These data indicate that extensive accumulation of neurofilaments in motor neurons can trigger a neurodegenerative process and may be a key intermediate in the pathway of pathogenesis leading to neuronal loss.

MDCK (epithelial cells from the dog kidney) plated at confluence, establish tight junctions in 12-15 hours through a process that requires protein synthesis, formation of a ring of actin filaments in close contact with the lateral membrane of the cells, calmodulin, and a Ca(2+)-dependent exocytic fusion of tight junction (TJ)-associated components. Monolayers incubated in the absence Ca2+ make no TJs. Yet, if Ca2+ is added under these circumstances, TJs are made with a faster kinetics. Ca2+ is needed mainly at a site located on the outer side of the cell membrane, where it activates uvomorulin and triggers the participation of the cellular components mentioned above, via G-proteins associated with phospholipase C and protein kinase C. In principle, the sites of all these molecules and mechanisms involved in junction formation may be where a variety of agents (hormones, drugs, metabolites) act to produce epithelia with a transepithelial electrical resistance (TER) ranging from 10 to 10,000 omega.cm2. This range may be also due to a variety of substances found in serum and in urine, that increase the TER in a reversible and dose-dependent manner.

Cell plasma membranes appear to be composed of domains, patches whose composition and function differ from the average for an entire membrane surface. Proteins and lipids may be segregated into domains by different mechanisms. Some of these mechanisms are discussed, followed by a summary of the evidence for membrane domains obtained in my laboratory. This evidence is largely based on measurements of the lateral diffusion of membrane proteins and lipids. Recent new approaches to the interpretation of lateral diffusion measurements, consideration of so-called fractal or long time-tails promise to give new insights into the stability and lifetime of membrane domains.

The retinal pigment epithelium (RPE) is a monolayer of cuboidal cells that lies in close association with the rod and cone photoreceptors. This epithelium has diverse features, three of which are discussed in some detail in this review, namely the daily phagocytosis of rod and cone outer segment fragments that are shed from their distal ends; the uptake, processing, transport and release of vitamin A (retinol) and some of its visual cycle intermediates (retinoids); and some of the aspects of its apical and basolateral membrane polarity that are the reverse of most other epithelia. Phagocytosis takes place at the apical surface via membrane receptor-mediated processes that are not yet well defined. Retinol uptake occurs at both the basolateral and apical surfaces by what appear to be separate receptor-mediated processes. The release of a crucial retinoid, 11-cis retinaldehyde (11-cis retinal), occurs solely across the apical membrane. Delivery of retinol across the basolateral membrane is mediated by a retinol binding protein (RBP) that is secreted by the liver as a complex with retinol (vitamin A). Within the cell, retinol and its derivatives are solubilized by intracellular retinoid binding proteins that are selective for retinol (cellular retinol binding protein, CRBP) and 11-cis retinoids (cellular retinal binding protein, CRALBP). Release of 11-cis retinal across the apical membrane and re-uptake of retinol from the photoreceptors during the visual cycle is promoted by an intercellular retinoid binding protein (IRBP). Na,K-ATPase, the membrane-integrated enzyme required to set up the ion gradients that drive other ion transporters, is largely localized to the apical membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

The epidermis of the skin is a stratified squamous epithelium, which plays an important protective role. It manifests this role by building an extensive cytoskeletal architecture, the unique feature of which is the presence of keratin filaments. There are two major pairs of keratins in the epidermis: one pair is expressed in dividing cells and the other expressed in terminally differentiating cells. As such, keratins provide useful biochemical markers to explore the molecular mechanisms underlying the balance between growth and differentiation in the epidermis. Here, I review what is currently known about epidermal growth and differentiation, and how an understanding of keratin gene expression has been useful in elucidating regulatory pathways in the skin.