Hypoxia increases human keratinocyte motility on connective tissue.

Re-epithelialization of skin wounds depends upon the migration of keratinocytes from the cut margins of the wound and is enhanced when human keratinocytes are covered with occlusive dressings that induce hypoxia. In this study, two independent migration assays were used to compare cellular motility on connective tissue components under normoxic or hypoxic conditions. Human keratinocytes apposed to collagens or fibronectin exhibited increased motility when subjected to hypoxic (0.2 or 2% oxygen) conditions compared with normoxic (9 or 20% oxygen) conditions. When compared with normoxic cells, hypoxic keratinocytes exhibited increased expression and redistribution of the lamellipodia-associated proteins (ezrin, radixin, and moesin). Furthermore, hypoxic keratinocytes demonstrated decreased secretion of laminin-5, a laminin isoform known to inhibit keratinocyte motility. Hypoxia did not alter the number of integrin receptors on the cell surface, but did induce enhanced secretion of the 92-kD type IV collagenase. These data demonstrate that hypoxia promotes human keratinocyte motility on connective tissue. Hypoxia-driven motility is associated with increased expression of lamellipodia proteins, increased expression of collagenase and decreased expression of laminin-5, the locomotion brake for keratinocytes.

Moesin, ezrin, and p205 are actin-binding proteins associated with neutrophil plasma membranes.

Actin-binding proteins in bovine neutrophil plasma membranes were identified using blot overlays with 125I-labeled F-actin. Along with surface-biotinylated proteins, membranes were enriched in major actin-binding polypeptides of 78, 81, and 205 kDa. Binding was specific for F-actin because G-actin did not bind. Further, unlabeled F-actin blocked the binding of 125I-labeled F-actin whereas other acidic biopolymers were relatively ineffective. Binding also was specifically inhibited by myosin subfragment 1, but not by CapZ or plasma gelsolin, suggesting that the membrane proteins, like myosin, bind along the sides of the actin filaments. The 78- and 81-kDa polypeptides were identified as moesin and ezrin, respectively, by co-migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with antibodies specific for moesin and ezrin. Although not present in detectable amounts in bovine neutrophils, radixin (a third and closely related member of this gene family) also bound 125I-labeled F-actin on blot overlays. Experiments with full-length and truncated bacterial fusion proteins localized the actin-binding site in moesin to the extreme carboxy terminus, a highly conserved sequence. Immunofluorescence micrographs of permeabilized cells and cell "footprints" showed moesin co-localization with actin at the cytoplasmic surface of the plasma membrane, consistent with a role as a membrane-actin-linking protein.

Imaging of dynamic changes of the actin cytoskeleton in microextensions of live NIH3T3 cells with a GFP fusion of the F-actin binding domain of moesin.

BACKGROUND: The cell surface undergoes continuous change during cell movement. This is characterized by transient protrusion and partial or complete retraction of microspikes, filopodia, and lamellipodia. This requires a dynamic actin cytoskeleton, moesin, components of Rho-mediated signal pathways, rearrangement of membrane constituents and the formation of focal adhesion sites. While the immunofluorescence distribution of endogenous moesin is that of a membrane-bound molecule with marked enhancement in some but not all microextensions, the C-terminal fragment of moesin co-distributes with filamentous actin consistent with its actin-binding activity. By taking advantage of this property we studied the spontaneous protrusive activity of live NIH3T3 cells, expressing a fusion of GFP and the C-terminal domain of moesin. RESULTS: C-moesin-GFP localized to stress fibers and was enriched in actively protruding cellular regions such as filopodia or lamellipodia. This localization was reversibly affected by cytochalasin D. Multiple types of cytoskeletal rearrangements were observed that occurred independent of each other in adjacent regions of the cell surface. Assembly and disassembly of actin filaments occurred repeatedly within the same space and was correlated with either membrane protrusion and retraction, or no change in shape when microextensions were adherent. CONCLUSIONS: Shape alone provided an inadequate criterion for distinguishing between retraction fibers and advancing, retracting or stable filopodia. Fluorescence imaging of C-moesin-GFP, however, paralleled the rapid and dynamic changes of the actin cytoskeleton in microextensions. Regional regulatory control is implicated because opposite changes occurred in close proximity and presumably independent of each other. This new and sensitive tool should be useful for investigating mechanisms of localized actin dynamics in the cell cortex.

Early neurogenesis of the mouse olfactory nerve: Golgi and electron microscopic studies.

The early neurogenesis of the mouse olfactory nerve, from its exist at the nasal epithelium to its entrance into the embryonic telencephalon, has been investigated by using the rapid Golgi method and electron microscopy. Previously unrecognized anatomical and possible functional interrelationships between developing olfactory nerve axons and their sheath cells have been observed: 1) at their exit from sensory epithelium (nasal compartment), 2) at their contact with the CNS surface (intracranial compartment), and 3) at their entrance into the embryonic telencephalon (central nervous tissue compartment). Based on these observations the anatomy of the mouse olfactory nerve is herein redefined. Exiting olfactory nerve axons and sheath cells from the same regions of the nasal epithelium establish an early association which is maintained up to their terminal glomerular neuropile. No disruptions have been found in either the olfactory nerve axons or in the continuity of their sheath cells from exit at the nasal epithelium to entrance into the developing olfactory bulb. Corresponding olfactory nerve axons with their sheath cells enter together and become incorporated into the developing olfactory bulb as units. Consequently, the cellular envelope of the olfactory glomerulus must be composed of olfactory sheath cells rather than of glial (astroglial) cells from the CNS. With this simple anatomical arrangement, a topographic map of the sensory epithelium could be established progressively in the developing olfactory bulb. Eventually, "regenerating" olfactory nerve axons from different nasal regions could be guided by their specific sheath cell conduits toward their target glomeruli; hence, the olfactory message may be maintained undisturbed throughout the life span of the animal. In addition, olfactory nerve axons establish synaptic-like contacts with their corresponding sheath cells prior to or during the perforation of the CNS surface. Reciprocal recognition between corresponding axons and their sheath cells at this crucial stage in their neurogenesis may play a significant role in the establishment of their terminal glomerulus. This new concept of the anatomy of the mammalian olfactory nerve should provide insights helpful in clarifying some of the still-unresolved questions regarding the structural and functional organizations of this primitive system.

Subcellular localization of moesin in dynamic filopodia, retraction fibers, and other structures involved in substrate exploration, attachment, and cell-cell contacts.

Moesin, a member of the talin-4.1 superfamily, is a linking protein of the submembraneous cytoskeleton. It is expressed in variable amounts in cells of different phenotypes such as macrophages, lymphocytes, fibroblastic, endothelial, epithelial, and neuronal cell lines. In this report we show that moesin is not randomly distributed throughout the cortical cytoskeleton, but rather that it is concentrated in specialized microdomains. It is localized in the intracellular core of microextensions known as filopodia, microvilli, microspikes, and retraction fibers. This subcellular distribution follows closely the dynamic changes in cell shape that take place when cells attach, spread, and move spontaneously or in response to extracellular signals. This suggests a similar function for moesin in diverse cell types related to the dynamic restructuring of domains of the plasma membrane and underlying membrane skeleton. Support for this comes from studies on PC-12 cells, which respond to NGF by extending neurites and moesin is redistributed from a diffuse localization to growth cone filopodia. In fibroblastic (NIH3T3) or macrophage (RAW264.7) cell lines, moesin is found in filopodia appearing at random on the cell surface soon after the cells are placed in culture, begin to attach, and spread. In polarized epithelial cells (LLC-PK1), moesin is associated with peripheral filopodia and apical microvilli. The cellular microextensions containing moesin are devoid of microtubules, focal contact proteins such as vinculin, and cortical cytoskeletal elements such as protein 4.1, but they do contain varying amounts of actin microfilaments. This localization of moesin in microextensions is not influenced by cytochalasin B. Treatment of cells with phorbolester (PMA) causes rapid cell spreading, disappearance of filopodia and retraction fibers, and moesin does not accumulate in the actin-rich lamellae that form at the cellular edges. After removal of PMA, cells retract and moesin again becomes concentrated in filopodia and retraction fibers. These studies support the hypothesis that filopodia, retraction fibers, and other microextensions of the plasma membrane are unique cellular microdomains with characteristic submembraneous components. Moesin could be involved in the dynamic restructuring of such microdomains by regulating binding interactions between the plasma membrane and the actin cytoskeleton.

Phosphorylation of threonine 558 in the carboxyl-terminal actin-binding domain of moesin by thrombin activation of human platelets.

The phosphorylation and localization of the membrane-linking protein moesin was analyzed during early activation of platelets with thrombin. Activated platelets elaborate filopodia and spread to assume flat pancake-like shapes, and moesin is localized in filopodia and cell body. In resting platelets, approximately 25% of moesin molecules are phosphorylated as shown by metabolic labeling with 32P(i) and by isoelectric focusing. Within seconds after exposure to thrombin, phosphorylation increases, reaching a maximum of 35% labeled molecules by 1 min, followed by a decrease to a new basal level within 5 min. This modification affects a single residue, Thr558, which is located within or close to a binding site for F-actin. Rapid shifts (0-100%) in the number of phosphorylated molecules are observed in the presence of inhibitors of serine/threonine kinases and phosphatases. Inhibitors affecting tyrosine phosphorylation also modulate phosphorylation at this site suggesting that the enzymes involved in the modification of Thr558 are regulated by tyrosine phosphorylation. Platelets respond to both extremes of modification by forming extremely long filopodia and the absence of spreading on glass. Completely phosphorylated moesin is concentrated together with F-actin in the center of the cell. The rapid modification of moesin at or near its actin-binding domain suggests a model for regulated membrane-cytoskeleton interaction during cell activation.

Phosphorylation of 558T of moesin detected by site-specific antibodies in RAW264.7 macrophages.

To determine, whether 558Thr in the carboxyl-terminal domain of moesin is phosphorylated in cells other than platelets, rabbit phosphorylation state-specific antibodies were made to the chemically phosphorylated synthetic hexapeptide KYKpTLR of the moesin sequence, as well as to the unphosphorylated form. The affinity-purified antibody populations were specific for either the phosphorylated or the unmodified peptide conjugated to BSA. Site-specific phosphorylation of moesin is detected in RAW macrophages by Western blot analysis, and immunofluorescence studies demonstrate that phosphorylated moesin is localized in filopodial protrusions. After pretreatment with the phosphatase inhibitor calyculin A, a similar effect to that seen in platelets in found, namely a substantial increase in moesin phosphorylation at 558Thr and redistribution of phospho-moesin together with F-actin into one or more ring-like structures in the cytoplasm, presumably due to binding of phosphorylated moesin to F-actin.

Disruption of dynamic cell surface architecture of NIH3T3 fibroblasts by the N-terminal domains of moesin and ezrin: in vivo imaging with GFP fusion proteins.

Lamellipodia, filopodia, microspikes and retraction fibers are characteristic features of a dynamic and continuously changing cell surface architecture and moesin, ezrin and radixin are thought to function in these microextensions as reversible links between plasma membrane proteins and actin microfilaments. Full-length and truncated domains of the three proteins were fused to green fluorescent protein (GFP), expressed in NIH3T3 cells, and distribution and behaviour of cells were analysed by using digitally enhanced differential interference contrast (DIC) and fluorescence video microscopy. The amino-terminal (N-)domains of all three proteins localize to the plasma membrane and fluorescence recordings parallel the dynamic changes in cell surface morphology observed by DIC microscopy of cultured cells. Expression of this domain, however, significantly affects cell surface architecture by the formation of abnormally long and fragile filopodia that poorly attach and retract abnormally. Even more striking are abundant irregular, branched and motionless membraneous structures that accumulate during retraction of lamellipodia. These are devoid of actin, endogenous moesin, ezrin and radixin, but contain the GFP-labeled domain. While a large proportion of endogenous proteins can be extracted with non-ionic detergents as in untransfected control cells, >90% of N-moesin and >60% of N-ezrin and N-radixin remain insoluble. The minimal size of the domain of moesin required for membrane localization and change in behavior includes residues 1-320. Deletions of amino acid residues from either end result in diffuse intracellular distribution, but also in normal cell behavior. Expression of GFP-fusions of full-length moesin or its carboxy-terminal domain has no effect on cell behavior during the observation period of 6-8 hours. The data suggest that, in the absence of the carboxy-terminal domain, N-moesin, -ezrin and -radixin interact tightly with the plasma membrane and interfere with normal functions of endogeneous proteins mainly during retraction.

The plasma membrane-actin linking protein, ezrin, is a glomerular epithelial cell marker in glomerulogenesis, in the adult kidney and in glomerular injury.

BACKGROUND: Ezrin belongs to a family of plasma membrane-cytoskeleton linking, actin binding proteins (Ezrin-radixin-Moesin family) involved in signal transduction, growth control, cell-cell adhesion, and microvilli formation. METHODS: The expression of ezrin was examined in glomerular cells in culture, during kidney development, in the mature kidney, and in five different experimental kidney disease models in the rat. RESULTS: Ezrin was specifically expressed in glomerular epithelial cells in developing glomeruli in mature glomeruli and in glomerular epithelial cells in culture. Distinct from its other family members, moesin and radixin, which are predominantly expressed in glomerular endothelial and mesangial areas, ezrin protein (by immunohistochemistry) was specifically and exclusively modulated during podocyte injury and regeneration. Ezrin immunohistochemistry was able to visualize cell body attenuation, pseudocysts, and in particular vacuolation of injured podocytes, a feature that usually has to be identified at the ultrastructural level, and was strikingly increased in binucleated podocytes or podocytes that were partially or completely detached from the underlying GBM (frequently also binucleated). Infiltrating macrophages also express ezrin, but can easily be differentiated from podocytes by their round shape and higher level of expression. CONCLUSIONS: Ezrin likely has a role in the cytoskeletal organization, such as reassembling of acting filaments accompanying podocyte injury and regeneration. Since suitable light microscopic markers for the identification of glomerular epithelial cells are rare, ezrin may also be a useful marker for podocytes in normal and injured glomeruli.