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J A Hammer

Publications and source records attributed to J A Hammer.

At least 37 records · Page 2Linked to original sources
PubMed1997

The predominant defect in dilute melanocytes is in melanosome distribution and not cell shape, supporting a role for myosin V in melanosome transport.

Mice with mutations at the dilute locus, which encodes the heavy chain of a type V unconventional myosin, exhibit a reduction in coat colour intensity. This defect is thought to be caused by the absence in dilute melanocytes of the extensive dendritic arbor through which these cells normally deliver pigment-laden melanosomes to keratinocytes. The data on which this conclusion has been based can also be explained, however, by a defect in the outward transport of melanosomes within melanocytes of normal shape. To resolve this question, we compared the shape and pigment distribution within melanocytes present in primary cultures prepared from the epidermis of C57BL/6J pups that were either wild type (D/D) at dilute or homozygous for the dilute null allele d120J. These same comparisons were also performed on melanocytes in situ, where antibodies to the membrane tyrosine kinase receptor cKIT were used to visualize melanocyte cell shape independent of pigment distribution. Wild type melanocytes were found to be dendritic and to have melanosomes distributed throughout their dendrites both in vitro and in situ. Mutant melanocytes were also found to be dendritic in both cases, but their melanosomes were highly concentrated in the cell body and largely excluded from dendrites. We conclude, therefore, that the predominant defect in dilute melanocytes is in melanosome distribution, not cell shape. These results argue that the myosin V isoform encoded by the dilute locus functions in dendritic extensions to move melanosomes from their site of formation within the cell body to their site of intercellular transfer at dendritic tips. This conclusion is consistent with our recent demonstration by immunolocalization that the dilute myosin V isoform associates with melanosomes in mouse melanocytes.

Animals↗
PubMed1997

The amino acid sequence of the light chain of Acanthamoeba myosin IC.

The amino acid sequence of the light chain of Acanthamoeba myosin IC deduced from the cDNA sequence comprises 149 amino acids with a calculated molecular weight of 16,739. All but the 3 N-terminal residues were also determined by amino acid sequencing of the purified protein, which also showed the N-terminus to be blocked. Phylogenetic analysis shows Acanthamoeba myosin IC light chain to be more similar to the calmodulin subfamily of EF-hand calcium-modulated proteins than to the myosin II essential light chain or regulatory light chain subfamilies. In pairwise comparisons, the myosin IC light chain sequence is most similar to sequences of calmodulins (approximately 50% identical) and a squid calcium-binding protein (approximately 43% identical); the sequence is approximately 37% identical to the calcium-binding essential light chain of Physarum myosin II and approximately 30% identical to the essential light chain of Acanthamoeba myosin II, and the essential light chain and regulatory light chain of Dictyostelium myosin II. The sequence predicts four helix-loop-helix domains with possible calcium-binding sites in domains I and III, suggesting that calcium may affect the activity of this unconventional myosin. This is the first report of the sequence of an unconventional myosin light chain other than calmodulin.

Acanthamoeba↗
PubMed1997

Myosin V associates with melanosomes in mouse melanocytes: evidence that myosin V is an organelle motor.

Mice with mutations at the dilute locus exhibit a 'washed out' or 'diluted' coat color. The pigments that are responsible for the coloration of mammalian hair are produced by melanocytes within a specialized organelle, the melanosome. Each melanocyte is responsible for delivering melanosomes via its extensive dendritic arbor to numerous keratinocytes, which go on to form the pigmented hair shaft. In this study, we show by light immunofluorescence microscopy and immunoelectron microscopy that the myosin V isoform encoded by the dilute locus associates with melanosomes. This association, which was seen in all mouse melanocyte cell lines examined and with two independent myosin V antibodies, was evident not only within completely melanized cells, but also within cells undergoing the process of melanosome biogenesis, where coordinate changes in the distributions of a melanosome marker and myosin V were seen. To determine where myosin V, a known actin-based motor, might play a role in melanosome transport, we also examined the cellular distribution of F-actin. The only region where myosin V and F-actin were both concentrated was in dendrites and dendritic tips, which represent the sole destination for melanosomes and where they accumulate in cultured melanocytes. These results support the idea that myosin V serves as the motor for the outward movement of melanosomes within dendritic extensions, and, together with the available information regarding the phenotype of mutant melanocytes in vitro, argue that coat color dilution is caused by the absense of this myosin V-dependent melanosome transport system.

Actins↗
PubMed1996

Structural change of the endoplasmic reticulum during fertilization: evidence for loss of membrane continuity using the green fluorescent protein.

Green fluorescent protein (GFP) was targeted to the lumen of the endoplasmic reticulum (ER) of starfish eggs by injecting mRNA coding for a chimeric protein containing a signal sequence and the KDEL ER retention sequence. By confocal microscopy, the GFP chimeric protein was localized in intracellular cisternae (membrane sheets) and the nuclear envelope, showing that it had been successfully targeted to the ER. The labeling pattern closely resembled that produced by the fluorescent dicarbocyanine DiI, which has been used previously to label the ER (Jaffe and Terasaki, Dev. Biol. 164, 579-587, 1994). Eggs expressing the GFP chimera were used to examine whether there is a loss of ER continuity at fertilization. The time required for recovery of fluorescence after photobleaching for both the GFP chimera and DiI was much longer in eggs at 1 min postfertilization than in unfertilized eggs or in 20-min-postfertilized eggs. This result provides strong evidence for a transient loss of continuity of the ER associated with Ca release at fertilization.

Animals↗
PubMed1996

The sequence of the dictyostelium myo J heavy chain gene predicts a novel, dimeric, unconventional myosin with a heavy chain molecular mass of 258 kDa.

The complete sequence of the Dictyostelium myo J heavy chain gene has been determined from overlapping genomic clones. The gene spans approximately 7400 base pairs, is split by two small introns, and encodes a 2241-residue, 258-kDa heavy chain polypeptide that that is composed of an N-terminal 944-residue myosin head domain, a central 863-residue domain that is predicted to form an alpha helical coiled-coil containing six hinges, and a C-terminal 434-residue globular domain. The head domain is notable in that it contains a approximately 30 residue insert near the nucleotide binding pocket, and five potential calmodulin/myosin light chain binding sites at the head/tail junction. The existence within the Myo J tail domain of both an extensive coiled-coil structure and a large globular domain suggests that this myosin is dimeric and incapable of self-assembly into filaments. While these properties, as well as the overall predicted structure of the Myo J protein, are reminiscent of class V myosins, the sequence of the 434-residue globular tail piece of Myo J shows no similarity to that of either yeast or vertebrate myosins V. Consistent with this, phylogenetic analyses based on myosin head sequence comparisons do not classify Myo J as a type V myosin. These and other sequence comparisons indicate that Myo J and two as-yet-unclassified unconventional myosins from Arabidopsis represent members of the newest class within the myosin superfamily (class XI). Northern blots analyses suggest that Myo J may function predominantly in vegetative Dictyostelium cells. Finally, Southern blot analyses suggest that Dictyostelium possesses another myosin that is very closely related to Myo J.

Amino Acid Sequence↗
PubMed1996

Dictyostelium mutants lacking multiple classic myosin I isoforms reveal combinations of shared and distinct functions.

Dictyostelium cells that lack the myoB isoform were previously shown to exhibit reduced efficiencies of phagocytosis and chemotactic aggregation ("streaming") and to crawl at about half the speed of wild-type cells. Of the four other Dictyostelium myosin I isoforms identified to date, myoC and myoD are the most similar to myoB in terms of tail domain sequence. Furthermore, we show here that myoC, like myoB and myoD, is concentrated in actin-rich cortical regions like the leading edge of migrating cells. To look for evidence of functional overlap between these isoforms, we analyzed myoB, myoC, and myoD single mutants, myoB/myoD double mutants, and myoB/myoC/myoD triple mutants, which were created using a combination of gene targeting techniques and constitutive expression of antisense RNA. With regard to the speed of locomoting, aggregation-stage cells, of the three single mutants, only the myoB mutant was significantly slower. Moreover, double and triple mutants were only slightly slower than the myoB single mutant. Consistent with this, the protein level of myoB alone rises dramatically during early development, suggesting that a special demand is placed on this one isoform when cells become highly motile. We also found, however, that the absolute amount of myoB protein in aggregation-stage cells is much higher than that for myoC and myoD, suggesting that what appears to be a case of nonoverlapping function could be the result of large differences in the amounts of functionally overlapping isoforms. Streaming assays also suggest that myoC plays a significant role in some aspect of motility other than cell speed. With regard to phagocytosis, both myoB and myoC single mutants exhibited significant reductions in initial rate, suggesting that these two isoforms perform nonredundant roles in supporting the phagocytic process. In triple mutants these defects were not additive, however. Finally, because double and triple mutants exhibited significant and progressive decreases in doubling times, we also measured the kinetics of fluid phase endocytic flux (uptake, transit time, efflux). Not only do all three isoforms contribute to this process, but their contributions are synergistic. While these results, when taken together, refute the simple notion that these three "classic" myosin I isoforms perform exclusively identical functions, they do reveal that all three share in supporting at least one cellular process (endocytosis), and they identify several other processes (motility, streaming, and phagocytosis) that are supported to a significant extent by either individual isoforms or various combinations of them.

Animals↗
PubMed1996

Localization of Dictyostelium myoB and myoD to filopodia and cell-cell contact sites using isoform-specific antibodies.

To date, five myosin I heavy chain genes (myoA-E) have been sequenced in Dictyostelium. Among them, myoB, myoC and myoD possess tail domains that contain a putative membrane-binding domain, a nucleotide-insensitive actin-binding site, and an src homology (SH)-3 domain. In this study, we have determined the intracellular localizations of myoB and myoD by immunofluorescence using isoform-specific antibodies raised against bacterially expressed fusion proteins. MyoB is concentrated at the leading edges of lamellipodia and at sites of cell-cell contact in both stationary and aggregation stage cells. Based on its distinctive appearance, we have named the myosin I-rich, interdigitating cell-cell contact structure in the stationary stage cells "zipper". To analyze the staining of filopodia, we employed the ratio imaging technique. We find that myoB is present in filopodia in either a uniform or an apical staining pattern. Like myoB, myoD is concentrated in leading edges of lamellipodia and at sites of cell-cell contact in stationary stage cells. MyoD is also present in filopodia, although the intensity is weaker than that of myoB staining. Despite persistence of myoD protein in the cells, myoD largely disappears from lamellipodia and cell-cell contact regions in aggregation stage cells, suggesting the occurrence of a developmentally regulated relocalization to the cytoplasm. While these results, along with the striking similarity in their tail domain structures, suggest that myoB and myoD have overlapping functions, differences in their localization in developing cells indicate that they have unique functions as well.

Animals↗
PubMed1994

Differentiation of oligodendrocytes cultured from developing rat brain is enhanced by exogenous GM3 ganglioside.

Cultures consisting primarily of O-2A progenitor cells and immature oligodendrocytes with a few microglia and astrocytes were obtained by shaking primary cultures from neonatal rat brain after 12-14 days in vitro. Addition of 50 micrograms/ml exogenous Neu-NAc alpha 2-3Gal beta 1-4Glc beta 1-1'ceramide (GM3 ganglioside) to the cultures resulted in an increase in the number and thickness of cell processes that stained intensely for sulfatide and galactocerebroside (galC) in comparison to control cultures without added GM3. The treated cultures also contained fewer astrocytes than control cultures as revealed by immunostaining for glial fibrillary acidic protein (GFAP). Cells that immunostained for both GFAP and sulfatide/galC were very rare in control cultures but were frequently seen in the GM3-treated cultures, suggesting that these may represent cells changing their direction of differentiation away from type II astrocytes toward oligodendrocytes under the influence of GM3. These effects on the developing rat oligodendrocytes were specific for GM3 ganglioside and were not produced by adding GM1, GM2, GD3, or GD1a to the cultures. Lactosyl ceramide and neuraminyl lactose were also ineffective. When control cultures were initially plated on polylysine and incubated with [14C]galactose, GD3 was the principal labeled ganglioside. However, as the control cells differentiated over time in culture without the addition of exogenous GM3 and produced increasing amounts of myelin-related components, the incorporation of [14C]galactose into endogenous GM3 increased to become the predominant labeled ganglioside by 6 days after plating. Metabolic labeling of the GM3-treated oligodendrocytes with [14C]galactose revealed increased incorporation into galC and sulfatide in comparison to control cultures, but a decreased labeling of endogenous GM3. Similarly, incorporation of an amino acid precursor into the myelin-associated glycoprotein (MAG) was increased by GM3 treatment, but incorporation into myelin basic protein (MBP) was not affected. Although the overall effect of added GM3 was to decrease the phosphorylation of most proteins in the oligodendrocytes, including MBP, GM3 enhanced the phosphorylation of MAG. These findings indicate that GM3 ganglioside has an important role in the differentiation of cells of the O-2A lineage toward myelin production, since differentiation is associated with increased metabolic labeling of endogenous GM3 in control cultures and is enhanced by the addition of exogenous GM3.

Animals↗
PubMed1994

The actin binding site in the tail domain of Dictyostelium myosin IC (myoC) resides within the glycine- and proline-rich sequence (tail homology region 2).

The majority of protozoan myosins I possess tail domains composed of three distinct and conserved regions of sequence, referred to as tail homology regions 1, 2 and 3 (TH.1, TH.2 and TH.3). While the N-terminal approximately half of the tail (corresponding to TH.1) has been implicated in membrane binding, all or some portion of the C-terminal approximately half of the tail (corresponding to TH.2 plus TH.3) has been implicated in binding to F-actin in a nucleotide-insensitive fashion. Here we show, using fusion proteins containing portions of the Dictyostelium myosin IC (myoC) tail domain and F-actin sedimentation assays, that the ability of the myoC tail to bind to actin resides entirely within the glycine- and proline-rich TH.2 domain. The src-like TH.3 domain does not bind to actin, nor does it augment the binding properties of the TH.2 domain. In addition to defining more precisely the location of the actin binding site in the tail domain of a protozoan myosin I, these results have implications for the function of the src-like TH.3 domain in myosins I and other proteins.

Actins↗
PubMed1993

Immunoreactivity of PMP-22, P0, and other 19 to 28 kDa glycoproteins in peripheral nerve myelin of mammals and fish with HNK1 and related antibodies.

Mammalian peripheral nervous system (PNS) myelin contains several glycoproteins with molecular weights of 19 to 28 kDa, including the major 28 kDa P0 glycoprotein and a recently cloned protein called PMP-22. Some glycoproteins in this M(r) range in humans, cats and some other mammals react with HNK1, a mouse monoclonal antibody that identifies a carbohydrate epitope shared between the immune system and a number of adhesion proteins in the nervous system. A variety of antibodies to P0, PMP-22, and the carbohydrate determinants reacting with HNK1 were used to characterize immunochemically these 19 to 28 kDa glycoproteins of cat PNS myelin. The HNK1-reactive components include P0 and two slightly smaller 23 to 26 kDa proteins that are immunologically related to P0. However, HNK1 reacts most strongly with a lower molecular weight glycoprotein that does not react with the antibodies to P0 and was identified as PMP-22. Since the carbohydrate structure reacting with HNK1 is generally expressed on adhesion molecules, this result suggests that PMP-22 may function in cell-cell or membrane-membrane interactions. Furthermore, the related human anti-MAG monoclonal IgM antibodies from patients with neuropathy also react strongly with PMP-22, suggesting that it may be a target antigen in the pathogenesis of this disease. Purified PNS and CNS myelin from bony fish (toadfish and trout) were also shown to contain major glycoproteins, in the same 19 to 28 kDa M(r) range, that react very strongly with HNK1. It is shown that fish myelin has major proteins of this size that are immunologically and structurally related to mammalian P0, and it is demonstrated here that one of the strongly HNK1-positive proteins reacted well with an antiserum raised to bovine P0. The presence of high levels of the adhesion-related HNK1 epitope on these major myelin proteins of fish suggests that this carbohydrate structure may have played a role in the molecular evolution of myelin.

Animals↗
PubMed1993

Sequence, expression pattern, intracellular localization, and targeted disruption of the Dictyostelium myosin ID heavy chain isoform.

The complete sequence of the Dictyostelium myosin ID (DMID) heavy chain isoform has been determined from cDNA and genomic clones. Like the DMIB isoform characterized previously, the DMID isoform is up-regulated during starvation-induced chemotactic aggregation, and its 124-kDa heavy chain contains the tail domain sequences that correspond to both the membrane and second actin-binding sites. An antibody that is specific for the DMID isoform was found to stain the actin-rich pseudopods at the leading edge of migrating cells. Protein microsequencing data reveals that the myosin I isoform localized to leading edge pseudopods in a previous study (Fukui, Y., Lynch, T. J., Brzeska, H., and Korn, E. D. (1989) Nature 341, 328-331) was DMIB, indicating that DMID and DMIB also colocalize and that both should influence the dynamics of actin-rich cortical structures. This and other data indicate that the DMID and DMIB isoforms are closely related and are distinct from the DMIA and DMIE isoforms, which possess truncated tail domains and are not up-regulated during chemotactic aggregation. Cells in which the DMID gene was rendered nonfunctional by targeted gene disruption do not show obvious behavioral defects, suggesting that another myosin I isoform(s) (possibly DMIB) might compensate for DMID. Finally, Southern blot data indicate that Dictyostelium may contain as many as nine myosin I isoforms.

Amino Acid Sequence↗
PubMed1993

The Dictyostelium myosin IE heavy chain gene encodes a truncated isoform that lacks sequences corresponding to the actin binding site in the tail.

We have isolated cDNA and genomic clones which together span the entire coding sequence for the 114.8 kDa heavy chain of Dictyostelium myosin IE (DMIE). The deduced primary sequence reveals a pattern characteristic of all myosins I, i.e., a myosin-like globular head domain fused to a tail domain that shows no similarity to the coiled-coil rod-like tail of type II myosins. The approx. 35 kDa tail domain of DMIE shows some sequence similarity to the membrane interaction region of other myosins I (tail-homology-region 1; TH-1), but lacks completely the sequences that correspond to the second actin binding site (the glycine-, proline- and alanine-rich TH-2 region and the src-like TH-3 region). Therefore, DMIE more closely resembles DMIA (Titus et al. (1989) Cell Regul 1, 55-63), which is also truncated, than DMIB and DMID, both of which possess all three tail homology regions. The similarity between the DMIE and DMIA isoforms extends to their pattern of expression, in which the steady state level of transcript for both genes is highest in vegetative cells and falls gradually after five to ten hours of starvation-induced development. Together, these results have important implications for interpreting and prioritizing gene targeting experiments designed to identify the functions of myosins I in vivo.

Amino Acid Sequence↗
PubMed1991

Myosin IB null mutants of Dictyostelium exhibit abnormalities in motility.

Cellular and intracellular motility are compared between normal Dictyostelium amoebae and amoebae lacking myosin IB (DMIB-). DMIB- cells generate elongated cell shapes, form particulate-free pseudopodia filled with F-actin, and exhibit an anterior bias in pseudopod extension in a fashion similar to normal amoebae. DMIB- cells also exhibit a normal response to the addition of the chemoattractant cAMP, including a depression in cellular and intracellular particle velocity, depolymerization of F-actin in pseudopodia, and a concomitant increase in cortical F-actin. DMIB- cells do, however, form lateral pseudopodia roughly three times as frequently as normal cells, turn more often, and exhibit depressed average instantaneous cell velocity. DMIB- cells also exhibit a decrease in the average instantaneous velocity of intracellular particle movement and an increase in the degree of randomness in particle direction. These findings indicate that if there is functional substitution for myosin IB by other myosin I isoforms, it is at best only partial, with myosin IB being necessary for maintenance of the normal rate and persistence of cellular translocation, suppression of lateral pseudopod formation and subsequent turning, rapid intracellular particle motility, and the normal anterograde bias of intracellular particle movement. Furthermore, it is likely that the behavioral abnormalities observed here for DMIB- cells underlie the delay in the onset of chemotactic aggregation, the increase in the time required to complete streaming, and the abnormalities in morphogenesis exhibited by DMIB- cells.

Actins↗
PubMed1991

Novel myosins.

The traditional view of myosin, drawn from studies of myosins from striated muscles, is that of an elongated two-headed molecule that assembles into filaments. However, biochemical, molecular genetic and genetic studies have uncovered a host of ubiquitous single-headed nonfilamentous myosins known collectively as myosins I. All of the myosins I possess the myosin head domain, the motor portion of muscle myosins they have tail the filament-forming tail domain of muscle myosins they have tail domains that interact variously with membranes, actin and calmodulin. These alternative molecular interactions confer novel motile properties on myosins I, such as the ability to move membranes relative to actin and to move actin relative to actin without having to assemble into filaments. The numerous actin-based movements retained by cells lacking myosin II, the two-headed filamentous form of nonmuscle myosin, may be supported by myosins I.

Journal Article↗
PubMed1991

Exogenous GM3 ganglioside stimulates process formation and glycoprotein release by cultured bovine oligodendrocytes.

Isolated adult bovine oligodendrocytes maintained in vitro for 10 days were treated for 1 day with 50 micrograms/ml of GM3 ganglioside (NeuNac alpha 2-3Gal beta 1-4Glc beta 1-1'ceramide) in serum-free culture medium. The treated oligodendrocytes had significantly longer processes with more branching than control cells in the same medium without GM3. The treatment also stimulated the release of a series of 22-100-kDa, [3H]glucosamine-labeled glycoproteins into the culture medium. Treatment of oligodendrocytes maintained in vitro for 50 days with GM3 for 1 day resulted in a thickening of the processes and the appearance of many fine branches on existing processes as well as a similar stimulation of glycoprotein release into the medium.

Animals↗
PubMed1991

Molecular cloning of protozoan myosin I heavy chain genes.

Myosins I are ubiquitous, nonfilamentous, actin-based mechanoenzymes originally discovered in protozoa. The extensive in vitro biochemical studies of purified protozoan myosins I are now being complemented with in vivo studies using cloned myosin I heavy chain genes and gene targeting techniques. Here we review briefly the systems and methods being used in these efforts to dissect protozoan myosin I structure and function using molecular genetic approaches.

Acanthamoeba↗