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M S Mooseker

Publications and source records attributed to M S Mooseker.

At least 37 records · Page 2Linked to original sources
PubMed1999

Drosophila quail, a villin-related protein, bundles actin filaments in apoptotic nurse cells.

Drosophila Quail protein is required for the completion of fast cytoplasm transport from nurse cells to the oocyte, an event critical for the production of viable oocytes. The abundant network of cytoplasmic filamentous actin, established at the onset of fast transport, is absent in quail mutant egg chambers. Previously, we showed that Quail is a germline-specific protein with sequence homology to villin, a vertebrate actin-regulating protein. In this study, we combined biochemical experiments with observations in egg chambers to define more precisely the function of this protein in the regulation of actin-bundle assembly in nurse cells. We report that recombinant Quail can bind and bundle filamentous actin in vitro in a manner similar to villin at a physiological calcium concentration. In contrast to villin, Quail is unable to sever or cap filamentous actin, or to promote nucleation of new actin filaments at a high calcium concentration. Instead, Quail bundles the filaments regardless of the calcium concentration. In vivo, the assembly of nurse-cell actin bundles is accompanied by extensive perforation of the nurse-cell nuclear envelopes, and both of these phenomena are manifestations of nurse-cell apoptosis. To investigate whether free calcium levels are affected during apoptosis, we loaded egg chambers with the calcium indicator Indo-1. Our observations indicate a rise in free calcium in the nurse-cell cytoplasm coincident with the permeabilization of the nuclear envelopes. We also show that human villin expressed in the Drosophila germline could sense elevated cytoplasmic calcium; in nurse cells with reduced levels of Quail protein, villin interfered with actin-bundle stability. We conclude that Quail efficiently assembles actin filaments into bundles in nurse cells and maintains their stability under fluctuating free calcium levels. We also propose a developmental model for the fast phase of cytoplasm transport incorporating findings presented in this study.

Actin Cytoskeleton↗
PubMed1998

Vesicle-associated brain myosin-V can be activated to catalyze actin-based transport.

Myosin-V has been linked to actin-based organelle transport by a variety of genetic, biochemical and localization studies. However, it has yet to be determined whether myosin-V functions as an organelle motor. To further investigate this possibility, we conducted a biochemical and functional analysis of organelle-associated brain myosin-V. Using the initial fractionation steps of an established protocol for the purification of brain myosin-V, we isolated a population of brain microsomes that is approx. fivefold enriched for myosin-V, and is similarly enriched for synaptic vesicle proteins. As demonstrated by immunoelectron microscopy, myosin-V associates with 30-40% of the vesicles in this population. Although a majority of myosin-V-associated vesicles also label with the synaptic vesicle marker protein, SV2, less than half of the total SV2-positive vesicles label with myosin-V. The average size of myosin-V/SV2 double-labeled vesicles (90+/-45 nm) is larger than vesicles that label only with SV2 antibodies (60+/-30 nm). To determine if these vesicles are capable of actin-based transport, we used an in vitro actin filament motility assay in which vesicles were adsorbed to motility assay substrates. As isolated, the myosin-V-associated vesicle fraction was nonmotile. However, vesicles pre-treated with ice-cold 0.1% Triton X-100 supported actin filament motility at rates comparable to those on purified myosin-V. This dilute detergent treatment did not disrupt vesicle integrity. Furthermore, while this treatment removed over 80% of the total vesicle proteins, myosin-V remained tightly vesicle-associated. Finally, function-blocking antibodies against the myosin-V motor domain completely inhibited motility on these substrates. These studies provide direct evidence that vesicle-associated myosin-V is capable of actin transport, and suggest that the activity of myosin-V may be regulated by proteins or lipids on the vesicle surface.

Actins↗
PubMed1998

Unconventional myosins in cell movement, membrane traffic, and signal transduction.

In the past few years genetic, biochemical, and cytolocalization data have implicated members of the myosin superfamily of actin-based molecular motors in a variety of cellular functions including membrane trafficking, cell movements, and signal transduction. The importance of myosins is illustrated by the identification of myosin genes as targets for disease-causing mutations. The task at hand is to decipher how the multitude of myosins function at both the molecular and cellular level-a task facilitated by our understanding of myosin structure and function in muscle.

Animals↗
PubMed1998

Human brush border myosin-I and myosin-Ic expression in human intestine and Caco-2BBe cells.

The human intestinal cell line, Caco-2BBe, has been established as an excellent model system for analysis of the enterocyte cytoskeleton including that of the actin rich apical brush border. To facilitate its use for functional analysis of a major component of the brush border, brush border myosin-I, human cDNAs encoding the heavy chain of this class I myosin were isolated and sequenced. The identity of this myosin as human brush border myosin-I was verified based on similarity with other vertebrate sequences, as well as its expression profile at both the RNA and protein levels. Localization of the protein in human intestine along the crypt-villus axis closely resembles that previously determined for brush border myosin-I in chicken, and is quite distinct from that of myosin-Ic, another myosin-I expressed in human intestine and Caco-2BBe cells. In immature cells of the crypt, brush border myosin-I staining is low, and there is significant cytosolic and basolateral localization, while villus cells stain much more intensely, and the protein is primarily localized to the brush border. Localization of myosin-Ic is essentially the inverse of brush border myosin-I in that crypt cells exhibit higher levels of staining, while villus cells have very low levels of myosin-Ic. The expression of both myosins-I was also examined during cell-contact induced differentiation of Caco-2BBe cells where expression and changes in localization closely resemble those that accompany differentiation of enterocyte in vivo.

Amino Acid Sequence↗
PubMed1998

Molecular genetic dissection of mouse unconventional myosin-VA: head region mutations.

The mouse dilute (d) locus encodes unconventional myosin-VA (MyoVA). Mice carrying null alleles of dilute have a lightened coat color and die from a neurological disorder resembling ataxia and opisthotonus within three weeks of birth. Immunological and ultrastructural studies suggest that MyoVA is involved in the transport of melanosomes in melanocytes and smooth endoplasmic reticulum in cerebellar Purkinje cells. In studies described here, we have used an RT-PCR-based sequencing approach to identify the mutations responsible for 17 viable dilute alleles that vary in their effects on coat color and the nervous system. Seven of these mutations mapped to the MyoVA motor domain and are reported here. Crystallographic modeling and mutant expression studies were used to predict how these mutations might affect motor domain function and to attempt to correlate these effects with the mutant phenotype.

Alleles↗
PubMed1998

Molecular genetic dissection of mouse unconventional myosin-VA: tail region mutations.

We used an RT-PCR-based sequencing approach to identify the mutations responsible for 17 viable dilute alleles, a mouse-coat-color locus encoding unconventional myosin-VA. Ten of the mutations mapped to the MyoVA tail and are reported here. These mutations represent the first extensive collection of tail mutations reported for any unconventional mammalian myosin. They identify sequences important for tail function and identify domains potentially involved in cargo binding and/or proper folding of the MyoVA tail. Our results also provide support for the notion that different myosin tail isoforms produced by alternative splicing encode important cell-type-specific functions.

Animals↗
PubMed1998

Human myosin-IXb is a mechanochemically active motor and a GAP for rho.

The heavy chains of the class IX myosins, rat myr5 and human myosin-IXb, contain within their tail domains a region with sequence homology to GTPase activating proteins for the rho family of G proteins. Because low levels of myosin-IXb expression preclude purification by conventional means, we have employed an immunoadsorption strategy to purify myosin-IXb, enabling us to characterize the mechanochemical and rho-GTPase activation properties of the native protein. In this report we have examined the light chain content, actin binding properties, in vitro motility and rho-GTPase activity of human myosin-IXb purified from leukocytes. The results presented here indicate that myosin-IXb contains calmodulin as a light chain and that it binds to actin with high affinity in both the absence and presence of ATP. Myosin-IXb is an active motor which, like other calmodulin-containing myosins, exhibits maximal velocity of actin filaments (15 nm/second) in the absence of Ca2+. Native myosin-IXb exhibits GAP activity on rho. Class IX myosins may be an important link between rho and rho-dependent remodeling of the actin cytoskeleton.

Actins↗
PubMed1997

Unconventional myosins in inner-ear sensory epithelia.

To understand how cells differentially use the dozens of myosin isozymes present in each genome, we examined the distribution of four unconventional myosin isozymes in the inner ear, a tissue that is particularly reliant on actin-rich structures and unconventional myosin isozymes. Of the four isozymes, each from a different class, three are expressed in the hair cells of amphibia and mammals. In stereocilia, constructed of cross-linked F-actin filaments, myosin-Ibeta is found mostly near stereociliary tips, myosin-VI is largely absent, and myosin-VIIa colocalizes with crosslinks that connect adjacent stereocilia. In the cuticular plate, a meshwork of actin filaments, myosin-Ibeta is excluded, myosin-VI is concentrated, and modest amounts of myosin-VIIa are present. These three myosin isozymes are excluded from other actin-rich domains, including the circumferential actin belt and the cortical actin network. A member of a fourth class, myosin-V, is not expressed in hair cells but is present at high levels in afferent nerve cells that innervate hair cells. Substantial amounts of myosins-Ibeta, -VI, and -VIIa are located in a pericuticular necklace that is largely free of F-actin, squeezed between (but not associated with) actin of the cuticular plate and the circumferential belt. Our localization results suggest specific functions for three hair-cell myosin isozymes. As suggested previously, myosin-Ibeta probably plays a role in adaptation; concentration of myosin-VI in cuticular plates and association with stereociliary rootlets suggest that this isozyme participates in rigidly anchoring stereocilia; and finally, colocalization with cross-links between adjacent stereocilia indicates that myosin-VIIa is required for the structural integrity of hair bundles.

Animals↗
PubMed1997

The genomic structure of the gene defective in Usher syndrome type Ib (MYO7A).

Usher syndrome type Ib is a recessive autosomal disorder manifested by congenital deafness, vestibular dysfunction, and progressive retinal degeneration. Mutations in the human myosin VIIa gene (MYO7A) have been reported to cause Usher type Ib. Here we report the genomic organization of MYO7A. An STS content map was determined to discover the YAC clones that would cover the critical region for Usher syndrome type Ib. Three of the YACs (802A5, 966D6, and 965F10) were subcloned into cosmids and used to assemble a preliminary cosmid contig of the critical region. Part of the gene encoding human myosin VIIa was found in the preliminary cosmid contig. A cosmid, P1, PAC, and long PCR contig that contained the entire MYO7A gene was assembled. Primers were designed from the composite cDNA sequence and used to detect intron-exon junctions by directly sequencing cosmid, P1, PAC, and genomic PCR DNA. Alternatively spliced products were transcribed from the MYO7A gene: the largest transcript (7.4 kb) contains 49 exons. The MYO7A gene is relatively large, spanning approximately 120 kb of genomic DNA on chromosome 11q13.

Abnormalities, Multiple↗
PubMed1997

Effects of shaker-1 mutations on myosin-VIIa protein and mRNA expression.

Numerous mammalian diseases have been found to be due to mutations in components of the actin cytoskeleton. Recently, mutations in the gene for an unconventional myosin, myosin-VIIa, were found to be the basis for the deafness and vestibular dysfunction observed in shaker-1 (sh1) mice and for a human deafness-blindness syndrome, Usher syndrome type 1B. Seven alleles of sh1 mice were analyzed to assess the affects of different myosin-VIIa mutations on both gene expression and tissue function. Myosin-VIIa is expressed in the inner ear and the retina, as well as the kidney, lung, and testis. Northern blot analysis indicated that myosin-VIIa mRNA expression, size, and stability were unaffected in the seven sh1 alleles. Immunoblot analysis showed that all seven alleles expressed some full-length myosin-VIIa protein. The range of expression, however, ran from sh1 [original], which expressed wild-type levels of protein, to two strains, sh1(4494SB) and sh1(4626SB), which expressed less than 1% of the normal level of myosin-VIIa protein. For the three alleles of sh1 that have been characterized and that have mutations in the motor domain, sh1 [original], sh1(816SB) and sh1(6J), the level of protein expression observed in these sh1 alleles correlated well with the predicted effects of the mutations on motor function. No change in retinal or testicular structure was observed at the light microscopic level during the life span of the seven sh1 alleles. Myosin-VIIa protein, when detectable, was observed to locate properly in the sh1 mice. On the basis of these results, we propose that the mutations in myosin-VIIa in the sh1 alleles leads to both motor dysfunction and to a protein destabilization phenotype.

Abnormalities, Multiple↗
PubMed1997

The growing family of myosin motors and their role in neurons and sensory cells.

Biochemical and physiological evidence has suggested that myosins, both conventional and unconventional, are critical for neurosensory activities. In the past few years, this premise has been supported by genetic evidence that has shown that unconventional myosins are essential for the proper functioning of neurons, retina and the sensory cells of the inner ear.

Animals↗
PubMed1997

An actin-related protein in Drosophila colocalizes with heterochromatin protein 1 in pericentric heterochromatin.

The actin-related proteins have been identified by virtue of their sequence similarity to actin. While their structures are thought to be closely homologous to actin, they exhibit a far greater range of functional diversity. We have localized the Drosophila actin-related protein, Arp4, to the nucleus. It is most abundant during embryogenesis but is expressed at all developmental stages. Within the nucleus Arp4 is primarily localized to the centric heterochromatin. Polytene chromosome spreads indicate it is also present at much lower levels in numerous euchromatic bands. The only other protein in Drosophila reported to be primarily localized to centric heterochromatin in polytene nuclei is heterochromatin protein 1 (HP1), which genetic evidence has linked to heterochromatin-mediated gene silencing and alterations in chromatin structure. The relationship between Arp4 and heterochromatin protein 1 (HP1) was investigated by labeling embryos and larval tissues with antibodies to Arp4 and HP1. Arp4 and HP1 exhibit almost superimposable heterochromatin localization patterns, remain associated with the heterochromatin throughout prepupal development, and exhibit similar changes in localization during the cell cycle. Polytene chromosome spreads indicate that the set of euchromatic bands labeled by each antibody overlap but are not identical. Arp4 and HP1 in parallel undergo several shifts in their nuclear localization patterns during embryogenesis, shifts that correlate with developmental changes in nuclear functions. The significance of their colocalization was further tested by examining nuclei that express mutant forms of HP1. In these nuclei the localization patterns of HP1 and Arp4 are altered in parallel fashion. The morphological, developmental and genetic data suggest that, like HP1, Arp4 may have a role in heterochromatin functions.

Actins↗
PubMed1997

Actin-binding membrane proteins identified by F-actin blot overlays.

Actin and associated proteins at the cytoskeleton-plasma membrane interface stabilize the membrane bilayer, control cell shape, and delimit specialized membrane domains. To identify membrane proteins that bind directly to F-actin, we have developed a blot overlay assay with 125I-labeled F-actin. In the soil amoebae, Dictyostelium discoideum, the major proteins reactive in this assay are p30a, a 34-kD peripheral membrane protein that is concentrated in filopodia and at sites of cell-cell adhesion, and ponticulin, a 17-kD transmembrane glycoprotein required for efficient chemotaxis and for control of pseudopod dynamics. Proteins with apparent molecular masses of approximately 34- and approximately 17-kD also are observed on F-actin blot overlays of many mammalian cell lines. However, in mammalian cells, the most prominent F-actin binding proteins in this assay exhibit apparent molecular masses of 78-, 80-, 81-, approximately 120-, and 205-kD. Bovine neutrophils contain the 78-, 81-, and 205-kD proteins, all of which co-isolate with a plasma membrane-enriched fraction. We have previously identified the 78-, 80-, and 81-kD proteins as moesin, radixin, and ezrin, respectively. These proteins, which are members of the protein 4.1 superfamily, colocalize with actin in cell surface extensions and have been implicated in the protrusion of microvilli, filopodia, and membrane ruffles. The 205-kD protein (p205) appears to be absent from current databases, and its characteristics are still under investigation. We here report that the 120-kD protein is drebrin, a submembranous actin-binding protein originally identified as a developmentally regulated brain protein. Thus, it appears that F-actin blot overlays provide an efficient assay for simultaneous monitoring of a subset of F-actin binding proteins, including p30a, ponticulin, moesin, radixin, ezrin, p205, and drebrin.

3T3 Cells↗
PubMed1996

Mapping of unconventional myosins in mouse and human.

Myosins are molecular motors that move along filamentous actin. Seven classes of myosin are expressed in vertebrates: conventional myosin, or myosin-II, as well as the 6 unconventional myosin classes-I, -V, -VI, -VII, -IX, and -X. We have mapped in mouse 22 probes encompassing all known unconventional myosins and, as a result, have identified 16 potential unconventional myosin genes. These genes include 7 myosins-I, 2 myosins-V, 1 myosin-VI, 3 myosins-VII, 2 myosins-IX, and 1 myosin-X. The map location of 5 of these genes was identified in human chromosomes by fluorescence in situ hybridization.

Animals↗
PubMed1996

Molecular cloning and domain structure of human myosin-VIIa, the gene product defective in Usher syndrome 1B.

Myosin-VIIa is an unconventional myosin with relatively restricted expression. Cloned first from an intestinal epithelium cell line, it occurs most notably in the testis, in the receptor cells of the inner ear, and in the pigment epithelium of the retina. Defects in myosin-VIIa cause the shaker-1 phenotype in mice and Usher syndrome 1B in human, which are characterized by deafness, lack of vestibular function, and (in human) progressive retinal degeneration. Because the described cDNAs encode less than half of the protein predicted from immunoblots, we have cloned cDNAs encoding the rest of human myosin-VIIa. Two transcripts were found, one encoding the predicted 250-kDa protein and another encoding a shorter form. Both transcripts were found in highest abundance in testis, although the shorter transcript was much less abundant. Both could be detected in lymphocytes by RT-PCR. The myosin tail encoded by the long transcript includes a long repeat of approximately 460 amino acids. Each repeat contains a novel "MyTH4" domain similar to domains in three other myosins, and a domain similar to the membrane-associated portion of talin and other members of the band-4.1 family.

Alternative Splicing↗
PubMed1996

Function of myosin-V in filopodial extension of neuronal growth cones.

The molecular mechanisms underlying directed motility of growth cones have not been determined. The role of myosin-V, an unconventional myosin, in growth cone dynamics was examined by chromophore-assisted laser inactivation (CALI). CALI of purified chick brain myosin-V absorbed onto nitrocellulose-coated cover slips inhibited the ability of myosin-V to translocate actin filaments. CALI of myosin-V in growth cones of chick dorsal root ganglion neurons resulted in rapid filopodial retraction. The rate of filopodial extension was significantly decreased, whereas the rate of filopodial retraction was not affected, which suggests a specific role for myosin-V in filopodial extension.

Adenosine Triphosphate↗
PubMed1996

Enzymatic characterization and functional domain mapping of brain myosin-V.

The actin binding and ATPase properties, as well as the functional domain structure of chick brain myosin-V, a two-headed, unconventional myosin, is reported here. Compared to conventional myosin from skeletal muscle, brain myosin-V exhibits low K-EDTA- and Ca-ATPase activities (1.8 and 0.8 ATP/s per head). The physiologically relevant Mg-ATPase is also low (approximately 0.3 ATP/s), unless activated by the presence of both F-actin and Ca2+ (Vmax of 27 ATP/s). Ca2+ stimulates the actin-activated Mg-ATPase over a narrow concentration range between 1 and 3 microM. In the presence of saturating Ca2+ and 75 mM KCl, surprisingly low concentrations of F-actin activate the Mg-ATPase in a hyperbolic manner (KATPase of 1.3 microM). Brain myosin-V also binds with relatively high affinity (compared to other known myosins) to F-actin in the presence of ATP, as assayed by cosedimentation. Digestion of brain myosin-V with calpain yielded a 65-kDa head domain fragment that cosediments with actin in an ATP-sensitive manner and a 80-kDa tail fragment that does not interact with F-actin. The 80-kDa fragment results from cleavage one residue beyond the proline-, glutamate-, serine-, threonine-rich region. Our data indicate that the Mg-ATPase cycle of brain myosin-V is tightly regulated by Ca2+, probably via direct binding to the calmodulin light chains in the neck domain, which like brush border myosin-I, results in partial (approximately 30%) dissociation of the calmodulin associated with brain myosin-V. The effect of Ca2+ binding, which appears to relieve suppression by the neck domain, can be mimicked by calpain cleavage near the head/neck junction.

Actins↗