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M S Mooseker

Publications and source records attributed to M S Mooseker.

At least 73 records · Page 4Linked to original sources
PubMed1994

Porcine myosin-VI: characterization of a new mammalian unconventional myosin.

We have cloned a new mammalian unconventional myosin, porcine myosin-VI from the proximal tubule cell line, LLC-PK1 (CL4). Porcine myosin-VI is highly homologous to Drosophila 95F myosin heavy chain, and together these two myosins comprise a sixth class of myosin motors. Myosin-VI exhibits ATP-sensitive actin-binding activities characteristic of myosins, and it is associated with a calmodulin light chain. Within LLC-PK1 cells, myosin-VI is soluble and does not associate with the major actin-containing domains. Within the kidney, however, myosin-VI is associated with sedimentable structures and specifically locates to the actin- and membrane-rich apical brush border domain of the proximal tubule cells. This motor was not enriched within the glomerulus, capillaries, or distal tubules. Myosin-VI associates with the proximal tubule cytoskeleton in an ATP-sensitive fashion, suggesting that this motor is associated with the actin cytoskeleton within the proximal tubule cells. Given the difference in association of myosin-VI with the apical cytoskeleton between LLC-PK1 cells and adult kidney, it is likely that this cell line does not fully differentiate to form functional proximal tubule cells. Myosin-VI may require the presence of additional elements, only found in vivo in proximal tubule cells, to properly locate to the apical domain.

Actins↗
PubMed1994

Multiple unconventional myosin domains of the intestinal brush border cytoskeleton.

Representatives of class V and class VI unconventional myosins are identified as components of the intestinal brush border cytoskeleton. With brush border myosin-I and myosin-II, this brings to four the number of myosin classes associated with this one subcellular domain and represents the first characterization of four classes of myosins expressed in a single metazoan cell type. The distribution and cytoskeletal association of each myosin is distinct as assessed by both biochemical fractionation and immunofluorescence localization. Myosin-VI exists in both the microvillus and terminal web although the terminal web is the predominant site of concentration. Myosin-V is present in the terminal web and, most notably, at the distal ends of the microvilli, thus becoming the first actin-binding protein to be localized to this domain as assessed by both immunohistochemical and biochemical methods. In the undifferentiated enterocytes of the intestinal crypts, myosin-VI is expressed but not yet localized to the brush border, in contrast to myosin-V, which does demonstrate an apical distribution in these cells. An assessment of myosin abundance indicates that while myosin-II is the most abundant in the cell and in the brush border, brush border myosin-I is only slightly less abundant in contrast to myosins-V and -VI, both of which are two orders of magnitude less abundant than the others. Extraction studies indicate that of these four myosins, myosin-V is the most tightly associated with the brush border membrane, as detergent, in addition to ATP, is required for efficient solubilization.

Actins↗
PubMed1993

Brain myosin-V is a two-headed unconventional myosin with motor activity.

Chicken myosin-V is a member of a recently recognized class of myosins distinct from both the myosins-I and the myosins-II. We report here the purification, electron microscopic visualization, and motor properties of a protein of this class. Myosin-V molecules consist of two heads attached to an approximately 30 nm stalk that ends in a globular region of unknown function. Myosin-V binds to and decorates F-actin, has actin-activated magnesium-ATPase activity, and is a barbed-end-directed motor capable of moving actin filaments at rates of up to 400 nm/s. Myosin-V does not form filaments. Each myosin-V heavy chain is associated with approximately four calmodulin light chains as well as two less abundant proteins of 23 and 17 kd.

Actins↗
PubMed1993

In vitro motilities of the unconventional myosins, brush border myosin-I, and chick brain myosin-V exhibit assay-dependent differences in velocity.

Two types of in vitro motility assays are currently used for examining the mechanochemical properties of purified myosins. The Nitella bead movement assay (Sheetz and Spudich: Nature 303:31-35, 1983) allows determination of both velocity and directionality of movement, but is of limited utility because of the fragile nature of the dissected Nitella internodal cells. On the other hand, the sliding actin filament assay (Kron and Spudich: Proc. Natl. Acad. Sci. U.S.A. 83:6272-6276, 1986) is technically much simpler to perform than the Nitella assay, and is suitable for the study of numerous physiological parameters. As it is currently used, however, the sliding actin filament assay does not indicate the directionality of motor movement. Previous studies have demonstrated that the velocities of filament-forming conventional myosins-II from either muscle or nonmuscle cells are comparable in both motility assays (Umemoto and Sellers: J. Biol. Chem. 265:14864-14869, 1990). However, similar studies using unconventional myosins are lacking. In the present report we have compared the rates of two structurally distinct unconventional myosins: brush border (BB) myosin-I and chick brain (CB) myosin-V (p190-calmodulin), using the sliding actin filament and Nitella-based in vitro motility assays. These two unconventional myosins differ from conventional myosins in that they appear unable to associate into bipolar filaments, and have extended rod-like neck domains which bind multiple calmodulin light chains in a Ca(2+)-sensitive manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Cloning of a secretory gelsolin from Drosophila melanogaster.

Gelsolin is an actin-binding protein with the abilities to sever and cap the barbed end of actin filaments and to promote the assembly of monomeric actin. It has been identified in vertebrates both as a cytoplasmic protein and as a protein secreted into the blood plasma. Here we report the nucleic acid sequence of the full-length complementary DNA for a secretory form of gelsolin from Drosophila. The deduced amino acid sequence of 790 residues (M(r) = 87,669) contains a predicted signal peptide of 20 amino acid residues. Comparison of the Drosophila gelsolin sequence with other members of the gelsolin family of actin-binding proteins reveals the characteristic segmental repeat structure found in this class of proteins. A 42% identity is observed between Drosophila secretory gelsolin and human plasma gelsolin when their primary structures are compared. Northern blots resolve a single 3000 base message in third instar Drosophila larvae, a message that appears to be encoded by a single gene located at 82A, B on the right arm of the third chromosome.

Amino Acid Sequence↗
PubMed1993

A novel cytoskeletal structure involved in purse string wound closure and cell polarity maintenance.

The process of wound repair in monolayers of the intestinal epithelial cell line, Caco-2BBe, was analyzed by a combination of time-lapse differential interference contrast (DIC) video and immunofluorescence microscopy, and laser scanning confocal immunofluorescence microscopy (LSCIM). DIC video analysis revealed that stab wounds made in Caco-2BBe monolayers healed by two distinct processes: (a) Extension of lamellipodia into the wounds; and (b) Purse string closure of the wound by distinct arcs or rings formed by cells bordering the wound. The arcs and rings which effected purse string closure appeared sharp and sheer in DIC, spanned between two and eight individual cells along the wound border, and contracted in a concerted fashion. Immunofluorescence analysis of the wounds demonstrated that the arcs and rings contained striking accumulations of actin filaments, myosin-II, villin, and tropomyosin. In contrast, arcs and rings contained no apparent enrichment of microtubules, brush border myosin-I immunogens, or myosin-V. LSCIM analysis confirmed the localization of actin filaments, myosin-II, villin, and tropomyosin in arcs and rings at wound borders. ZO-1 (a tight junction protein), also accumulated in arcs and rings around wounds, despite the fact that cell-cell contacts are absent at wound borders. Sucrase-isomaltase, an apically-localized integral membrane protein, maintained an apical localization in cells where arcs or rings were formed, but was found in lamellipodia extending into wounds in cells where arcs failed to form. Time-course, LSCIM quantification of actin, myosin II, and ZO-1 revealed that accumulation of these proteins within arcs and rings at the wound edge began within 5 minutes and peaked within 30-60 minutes of wounding. Actin filaments, myosin-II, and ZO-1 achieved 10-, 3-, and 4-fold enrichments, respectively, relative to cell edges which did not border wounds. The results demonstrate that wounded Caco-2BBe monolayers assemble a novel cytoskeletal structure at the borders of wounds. The results further suggest that this structure plays at least two roles in wound repair; first, mediation of concerted, purse string movement of cells into the area of the wound and second, maintenance of apical/basolateral polarity in cells which border the wound.

Actins↗
PubMed1993

Calcium-calmodulin and regulation of brush border myosin-I MgATPase and mechanochemistry.

We examined the Ca(2+)-dependent regulation of brush border (BB) myosin-I by probing the possible roles of the calmodulin (CM) light chains. BB myosin-I MgATPase activity, sensitivity to chymotryptic digestion, and mechanochemical properties were assessed using 1-10 microM Ca2+ and in the presence of exogenously added CM since it has been proposed that this myosin is regulated by calcium-induced CM dissociation from the 119-kD heavy chain. Each of these BB myosin-I properties were dramatically altered by the same threshold of 2-3 microM Ca2+. Enzymatically active NH2-terminal proteolytic fragments of BB myosin-I which lack the CM binding domains (the 78-kD peptide) differ from CM-containing peptides in that the former is completely insensitive to Ca2+. Furthermore, the 78-kD peptide exhibits high levels of MgATPase activity which are comparable to that observed for BB myosin-I in the presence of Ca2+. This suggests that Ca2+ regulates BB myosin-I MgATPase by binding directly to the CM light chains, and that CM acts to repress endogenous MgATPase activity. Ca(2+)-induced CM dissociation from BB myosin-I can be prevented by the addition of exogenous CM. Under these conditions Ca2+ causes a reversible slowing of motility. In contrast, in the absence of exogenous CM, motility is stopped by Ca2+. We demonstrate this reversible slowing is not due to the presence of inactive BB myosin-I molecules exerting a "braking" effect on motile filaments. However, we did observe Ca(2+)-independent slowing of motility by acidic phospholipids, suggesting that factors other than Ca2+ and CM content can affect the mechanochemical properties of BB myosin-I.

Actin Cytoskeleton↗
PubMed1993

An in vitro model for the analysis of intestinal brush border assembly. I. Ultrastructural analysis of cell contact-induced brush border assembly in Caco-2BBe cells.

Intestinal epithelial cells assemble and maintain a polarized, highly organized membrane-cytoskeleton array, the brush border. We describe an in vitro, cell contact-induced brush border assembly model using the Caco-2BBe clones. Subconfluent cells were 'depolarized' by brief passage through suspension culture in the presence of cytochalasin D and re-plated on filters at high density in low-Ca2+ medium. Upon return to regular medium, these small, rounded cells with bleb-like protrusions formed, over the course of 19 days, a polarized monolayer of tall, columnar cells with a well-defined brush border. Ultrastructural changes were documented by both transmission and scanning electron microscopy. The earliest events of microvillar assembly coincided with a short period of cell aggregation. Intercellular cysts were occasionally observed within these aggregates, and junction formation between cells which had no contact with the filter was also observed. Monolayer formation was completed within 48 hours, and cell height steadily increased approximately 3.5-fold over 19 days. Concurrent with monolayer formation and the increase in cell height, sparse microvilli with a few actin core filaments gradually became more dense and better organized. By the third day, the actin core bundles had begun to extend into the subjacent cytoplasm, while terminal web assembly was underway at five days. The mature morphology of the brush border was first observed at nine days, although cell height and microvillar density continued to increase during the subsequent ten days. Microvillar density rose approximately nine-fold throughout brush border assembly in the Caco-2BBe cells. With the exception of the formation of cellular aggregates at the onset of the time course, this sequence of morphological changes is comparable to that observed during brush border assembly in embryonic intestinal epithelial cells. The Caco-2BBe assembly model provides a useful system in which to investigate various molecular aspects of brush border assembly.

Actins↗
PubMed1993

An in vitro model for the analysis of intestinal brush border assembly. II. Changes in expression and localization of brush border proteins during cell contact-induced brush border assembly in Caco-2BBe cells.

In the companion paper (M. D. Peterson and M. S. Mooseker (1993). J. Cell Sci. 105, 445-460) we describe a method for modeling brush border assembly in the Caco-2BBe clones. In this study we have examined the molecular changes accompanying cell contact-induced brush border assembly. A subset of brush border proteins was tracked throughout brush border assembly by immunoblotting and by immunofluorescent localization using laser scanning confocal microscopy. Actin, fodrin, villin and presumptive unconventional myosin immunogens were distributed at the periphery of depolarized cells. All proteins partitioned primarily with the membrane fraction upon differential sedimentation of depolarized cell lysates; the fractionation patterns were comparable to those of confluent cells. After a monolayer had formed, each protein showed a redistribution to the apical domain in a discrete sequence. Actin and villin began to shift apically at 2 d, while fodrin and the unconventional myosin immunogens did not redistribute until 3 d. Enterocyte-like localization was observed by 5 d for all proteins. Sucrase-isomaltase was not reliably detectable until 9 d by immunofluorescence, after brush border assembly was complete. Quantitative immunoblot analysis of total cell extracts demonstrated an average 10-fold increase in villin levels, while fodrin levels appeared to remain unchanged. Three putative unconventional myosin immunogens of 140 kDa, 130 kDa, and 110 kDa have been detected previously in the C2BBe cells with a head-specific monoclonal antibody to avian brush border myosin I (M. D. Peterson and M. S. Mooseker (1992) J. Cell Sci. 102, 581-600). Each of these immunogens displayed distinct expression patterns during brush border assembly. The 140 kDa species decreased by half, while the 130 kDa immunogen(s) did not change in any consistent fashion. The 110 kDa protein, presumed to be human brush border myosin I, rose on average 8-fold. A ribonuclease protection assay was also performed using a probe for human brush border myosin I. Equal amounts of total RNA from depolarized and confluent cells were assayed; the level of protected product was approximately 9-fold greater in the confluent cells. The expression patterns of the brush border proteins, coupled with the correlation to the ultrastructural features during brush border assembly in C2BBe cells, show that differentiation of the C2BBe cells closely resembles the changes that occur during human fetal intestinal differentiation.

Actins↗
PubMed1992

Unconventional myosins.

The unconventional myosins form a large and diverse group of molecular motors. The number of known unconventional myosins is increasing rapidly and in the past year alone two new classes have been identified. Substantial progress has been made towards characterizing the properties and functions of these motor proteins, which have been hypothesized to play fundamental roles in processes such as cell locomotion, phagocytosis and vesicle transport.

Acanthamoeba↗
PubMed1992

Localization of capping protein in chicken epithelial cells by immunofluorescence and biochemical fractionation.

We have localized capping protein in epithelial cells of several chicken tissues using affinity-purified polyclonal antibodies and immunofluorescence. Capping protein has a distribution in each tissue coincident with proteins of the cell-cell junctional complex, which includes the zonula adherens, zonula occludens, and desmosome. "En face" views of the epithelial cells showed capping protein distributed in a polygonal pattern coincident with cell boundaries in intestinal epithelium, sensory epithelium of the cochlea, and the pigmented epithelium of the retina and at regions of cell-cell contact between chick embryo kidney cells in culture. "Edge-on" views obtained by confocal microscopy of intact single intestinal epithelial cells and of retinal pigmented epithelium showed that capping protein is located in the apical region of the epithelial cells coincident with the junctional complexes. These images do not resolve the individual types of junctions of the junctional complex. Immunolabeling of microvilli or stereocilia was faint or not detectable. Capping protein was also detected in the cytoplasm of intact intestinal epithelial cells and in nuclei of cells in the pigmented retina and in the kidney cell cultures, but not in nuclei of cells of the intestinal epithelium or sensory epithelium. Biochemical fractionation of isolated intestinal epithelial cells shows capping protein in the brush border fraction, which contains the junctional complexes, and in the soluble fraction. These results are consistent with the results of the immunolabeling experiments. Highly purified microvilli of the brush borders also contained capping protein; this result was unexpected based on the low intensity of immunofluorescence staining of microvilli and stereocilia. The microvilli were not contaminated with junctional complexes, as defined by the absence of several markers for cell junctions. The cause and significance of this discrepancy is not certain at this time. Since capping protein binds the barbed end of actin filaments in vitro, we hypothesize that capping protein is bound to the barbed ends of actin filaments associated with one or more of the junctions of the junctional complex.

Actin Depolymerizing Factors↗
PubMed1992

Biochemical and immunological characterization of p190-calmodulin complex from vertebrate brain: a novel calmodulin-binding myosin.

We have recently identified a novel 190-kD calmodulin-binding protein (p190) associated with the actin-based cytoskeleton from mammalian brain (Larson, R. E., D. E. Pitta, and J. A. Ferro. 1988. Braz. J. Med. Biol. Res. 21:213-217; Larson, R. E., F. S. Espindola, and E. M. Espreafico. 1990. J. Neurochem. 54:1288-1294). These studies indicated that p190 is a phosphoprotein substrate for calmodulin-dependent kinase II and has calcium- and calmodulin-stimulated MgATPase activity. We now have biochemical and immunological evidence that this protein is a novel calmodulin-binding myosin whose properties include (a) Ca2+ dependent action activation of its Mg-ATPase activity, which seems to be mediated by Ca2+ binding directly to calmodulin(s) associated with p190 (maximal activation by actin requires the presence of Ca2+ and is further augmented by addition of exogenous calmodulin); (b) ATP-sensitive cross-linking of skeletal muscle F-actin, as demonstrated by the low-speed actin sedimentation assay; and (c) cross-reactivity with mAbs specific for epitopes in the head of brush border myosin I. We also show that p190 has properties distinct from conventional brain myosin II and brush border myosin I, including (a) separation of p190 from brain myosin II by gel filtration on a Sephacryl S-500 column; (b) lack by p190 of K(+)-stimulated EDTA ATPase activity characteristic of most myosins; (c) lack of immunological cross-reactivity of polyclonal antibodies which recognize p190 and brain myosin II, respectively; (d) lack of immunological recognition of p190 by mAbs against an epitope in the tail region of brush border myosin I; and (e) distinctive proteolytic susceptibility to calpain. A survey of rat tissues by immunoblotting indicated that p190 is expressed predominantly in the adult forebrain and cerebellum, and could be detected in embryos 11 d post coitus. Immunocytochemical studies showed p190 to be present in the perikarya and dendritic extensions of Purkinje cells of the cerebellum.

Adenosine Triphosphatases↗
PubMed1992

Primary structure and cellular localization of chicken brain myosin-V (p190), an unconventional myosin with calmodulin light chains.

Recent biochemical studies of p190, a calmodulin (CM)-binding protein purified from vertebrate brain, have demonstrated that this protein, purified as a complex with bound CM, shares a number of properties with myosins (Espindola, F. S., E. M. Espreafico, M. V. Coelho, A. R. Martins, F. R. C. Costa, M. S. Mooseker, and R. E. Larson. 1992. J. Cell Biol. 118:359-368). To determine whether or not p190 was a member of the myosin family of proteins, a set of overlapping cDNAs encoding the full-length protein sequence of chicken brain p190 was isolated and sequenced. Verification that the deduced primary structure was that of p190 was demonstrated through microsequence analysis of a cyanogen bromide peptide generated from chick brain p190. The deduced primary structure of chicken brain p190 revealed that this 1,830-amino acid (aa) 212,509-D) protein is a member of a novel structural class of unconventional myosins that includes the gene products encoded by the dilute locus of mouse and the MYO2 gene of Saccharomyces cerevisiae. We have named the p190-CM complex "myosin-V" based on the results of a detailed sequence comparison of the head domains of 29 myosin heavy chains (hc), which has revealed that this myosin, based on head structure, is the fifth of six distinct structural classes of myosin to be described thus far. Like the presumed products of the mouse dilute and yeast MYO2 genes, the head domain of chicken myosin-V hc (aa 1-764) is linked to a "neck" domain (aa 765-909) consisting of six tandem repeats of an approximately 23-aa "IQ-motif." All known myosins contain at least one such motif at their head-tail junctions; these IQ-motifs may function as calmodulin or light chain binding sites. The tail domain of chicken myosin-V consists of an initial 511 aa predicted to form several segments of coiled-coil alpha helix followed by a terminal 410-aa globular domain (aa, 1,421-1,830). Interestingly, a portion of the tail domain (aa, 1,094-1,830) shares 58% amino acid sequence identity with a 723-aa protein from mouse brain reported to be a glutamic acid decarboxylase. The neck region of chicken myosin-V, which contains the IQ-motifs, was demonstrated to contain the binding sites for CM by analyzing CM binding to bacterially expressed fusion proteins containing the head, neck, and tail domains. Immunolocalization of myosin-V in brain and in cultured cells revealed an unusual distribution for this myosin in both neurons and nonneuronal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

Characterization of the enterocyte-like brush border cytoskeleton of the C2BBe clones of the human intestinal cell line, Caco-2.

The brush border (BB) of the enterocyte is a well-studied example of the actin-based cytoskeleton. We describe here a cell culture model that expresses a faithful representation of the in vivo structure. Two clones (C2BBe 1 and 2) isolated from the cell line Caco-2 (derived from a human colonic adenocarcinoma) formed a polarized monolayer with an apical BB morphologically comparable to that of the human colon. BBs could be isolated by standard methods and contained the microvillar proteins villin, fimbrin, sucrase-isomaltase and BB myosin I, and the terminal web proteins fodrin and myosin II. The immunolocalization of these proteins in confluent, filter-grown monolayers was determined by laser scanning confocal microscopy; patterns of distribution comparable to those in human enterocytes were observed. Sedimentation analysis of cell homogenates derived from C2BBe cells and human colonic epithelial cells demonstrated similar patterns of fractionation of BB proteins; the physical association of those proteins, as determined by extraction from the BB, was also comparable between the two cell types. Like enterocytes of the human intestine, C2BBe cells expressed multiple myosin I immunogens reactive with a head domain-specific monoclonal antibody raised against avian BB myosin I, one of which co-migrated with the approximately 110 kilodalton (kDa) heavy chain of human BB myosin I. In addition, the C2BBe cells express a pair of higher molecular mass immunogens (130 and 140 kDa). These myosin I immunogens all exhibit ATP-dependent association with the C2BBe cytoskeleton. Although the higher molecular mass immunogens were detected in several other human intestinal lines examined, including the parent Caco-2 line, none of these other lines expressed detectable levels of the 110 kDa immunogen, which is presumed to be the heavy chain of human BB myosin I.

Cell Line↗
PubMed1992

The molecular architecture of an insect midgut brush border cytoskeleton.

The cytoskeletal apparatus of the vertebrate intestinal brush border (BB) has served as a model system for the actin-based cytoskeleton of nonmuscle cells. In this study, we examine the structural organization and molecular architecture of the BB cytoskeleton expressed in the midgut of lepidopteran larvae, Manduca sexta. Electron microscopy of the midgut of the 5th instar larvae revealed enterocytes with an apical BB surface comparable to that in the vertebrate intestine, with both microvillar (MV) and terminal web (TW) domains, the latter defined by a zone of organelle exclusion directly beneath the MV. As reported previously for the larval dragon fly, the MV contain a bundle of actin filaments, as determined by staining with rhodamine phalloidin (Kukulies, J., et al., Protoplasma 121, 157-162 (1984)) and heavy meromyosin decoration (Komnick, H., J. Kukulies, Zoomorphology 107, 241-253 (1987)). Two-dimensional gel analysis revealed the presence of multiple isoelectric variants of actin with the major isoform corresponding to the non-muscle actin isoform II, expressed in Drosophila. Like the vertebrate BB, the Manduca BB can be isolated intact from enterocytes by mechanical shear. Immunochemical analysis of isolated BB fractions or whole homogenates of midgut revealed proteins of appropriate molecular weight immunoreactive with antibodies to the MV core proteins: BB myosin I, villin and fimbrin, and the TW components: spectrin, myosin II and tropomyosin. Immunocytochemical localization of a subset of these proteins at the light microscopic (spectrin) and electron microscopic (actin, villin, spectrin, myosin II, and tropomyosin) level reveals that the molecular architecture of the Manduca BB cytoskeleton is homologous to that found in vertebrates.

Animals↗