Myosins.

The number and variety of myosins that have been identified has increased greatly over the past few years, and is still growing. Myosins have been classified into at least six distinct classes. Research during the last year has concentrated on identifying the roles of various myosins.

Myosins: matching functions with motors.

It is an exciting time to be studying myosins and their roles in the function of cells and organisms. Past efforts aimed at finding new members of this family have now given way to a focus on identifying individual functions for each motor protein. These actin-based motors are now known to be intimately involved in the following processes: neurosensory function; vesicle trafficking; determinant partitioning; and cortical function. The following article reviews the inroads made into the functions of myosins in these processes over the past several years.

VLBI measurement of the vector baseline between geodetic antennas at Kokee Park Geophysical Observatory, Hawaii.

We measured the components of the 31-m-long vector between the two very-long-baseline interferometry (VLBI) antennas at the Kokee Park Geophysical Observatory (KPGO), Hawaii, with approximately 1 mm precision using phase delay observables from dedicated VLBI observations in 2016 and 2018. The two KPGO antennas are the 20 m legacy VLBI antenna and the 12 m VLBI Global Observing System (VGOS) antenna. Independent estimates of the vector between the two antennas were obtained by the National Geodetic Survey (NGS) using standard optical surveys in 2015 and 2018. The uncertainties of the latter survey were 0.3 and 0.7 mm in the horizontal and vertical components of the baseline, respectively. We applied corrections to the measured positions for the varying thermal deformation of the antennas on the different days of the VLBI and survey measurements, which can amount to 1 mm, bringing all results to a common reference temperature. The difference between the VLBI and survey results are 0.2 ± 0.4 mm, -1.3 ± 0.4 mm, and 0.8 ± 0.8 mm in the East, North, and Up topocentric components, respectively. We also estimate that the Up component of the baseline may suffer from systematic errors due to gravitational deformation and uncalibrated instrumental delay variations at the 20 m antenna that may reach ± 10 and -2 mm, respectively, resulting in an accuracy uncertainty on the order of 10 mm for the relative heights of the antennas. Furthermore, possible tilting of the 12 m antenna increases the uncertainties in the differences in the horizontal components to 1.0 mm. These results bring into focus the importance of (1) correcting to a common reference temperature the measurements of the reference points of all geodetic instruments within a site, (2) obtaining measurements of the gravitational deformation of all antennas, and (3) monitoring local motions of the geodetic instruments. These results have significant implications for the accuracy of global reference frames that require accurate local ties between geodetic instruments, such as the International Terrestrial Reference Frame (ITRF).

Unconventional myosins: new frontiers in actin-based motors.

The unconventional myosins are a superfamily of actin-based motors responsible for a rich array of intracellular motility events. Recent evidence suggests that these motors play important roles in cell migration, endocytosis and intracellular transport. Several genetic mutants have been identified whose abnormalities are the result of the loss of a specific myosin. This article describes how analysis of these mutants, coupled with basic studies of the intracellular localization and biochemical properties of individual myosins, is leading to a clearer understanding of the in vivo function of a number of these interesting motor proteins.

Myosin I overexpression impairs cell migration.

Dictyostelium myoB, a member of the myosin I family of motor proteins, is important for controlling the formation and retraction of membrane projections by the cell's actin cortex (Novak, K.D., M.D. Peterson, M.C. Reedy, and M.A. Titus. 1995. J. Cell Biol. 131:1205-1221). Mutants that express a three- to sevenfold excess of myoB (myoB+ cells) were generated to further analyze the role of myosin I in these processes. The myoB+ cells move with an instantaneous velocity that is 35% of the wild-type rate and exhibit a 6-8-h delay in initiation of aggregation when placed under starvation conditions. The myoB+ cells complete the developmental cycle after an extended period of time, but they form fewer fruiting bodies that appear to be small and abnormal. The myoB+ cells are also deficient in their ability both to form distinct F-actin filled projections such as crowns and to become elongate and polarized. This defect can be attributed to the presence of at least threefold more myoB at the cortex of the myoB+ cells. In contrast, threefold overexpression of a truncated myoB that lacks the src homology 3 (SH3) domain (myoB/SH3- cells) or myoB in which the consensus heavy chain phosphorylation site was mutated to an alanine (S332A-myoB) does not disturb normal cellular function. However, there is an increased concentration of myoB in the cortex of the myoB/SH3- and S332A-myoB cells comparable to that found in the myoB+ cells. These results suggest that excess full-length cortical myoB prevents the formation of the actin-filled extensions required for locomotion by increasing the tension of the F-actin cytoskeleton and/or retracting projections before they can fully extend. They also demonstrate a role for the phosphorylation site and SH3 domain in mediating the in vivo activity of myosin I.

Dictyostelium myosin I double mutants exhibit conditional defects in pinocytosis.

The functional relationship between three Dictyostelium myosin Is, myoA, myoB, and myoC, has been examined through the creation of double mutants. Two double mutants, myoA-/B- and myoB-/C-, exhibit similar conditional defects in fluid-phase pinocytosis. Double mutants grown in suspension culture are significantly impaired in their ability to take in nutrients from the medium, whereas they are almost indistinguishable from wild-type and single mutant strains when grown on a surface. The double mutants are also found to internalize gp126, a 116-kD membrane protein, at a slower rate than either the wild-type or single mutant cells. Ultrastructural analysis reveals that both double mutants possess numerous small vesicles, in contrast to the wild-type or myosin I single mutants that exhibit several large, clear vacuoles. The alterations in fluid and membrane internalization in the suspension-grown double mutants, coupled with the altered vesicular profile, suggest that these cells may be compromised during the early stages of pinocytosis, a process that has been proposed to occur via actin-based cytoskeletal rearrangements. Scanning electron microscopy and rhodamine-phalloidin staining indicates that the myosin I double mutants appear to extend a larger number of actin-filled structures, such as filopodia and crowns, than wild-type cells. Rhodamine-phalloidin staining of the F-actin cytoskeleton of these suspension-grown cells also reveals that the double mutant cells are delayed in the rearrangement of cortical actin-rich structures upon adhesion to a substrate. We propose that myoA, myoB, and myoC play roles in controlling F-actin filled membrane projections that are required for pinosome internalization in suspension.

Myosin-I nomenclature.

We suggest that the vertebrate myosin-I field adopt a common nomenclature system based on the names adopted by the Human Genome Organization (HUGO). At present, the myosin-I nomenclature is very confusing; not only are several systems in use, but several different genes have been given the same name. Despite their faults, we believe that the names adopted by the HUGO nomenclature group for genome annotation are the best compromise, and we recommend universal adoption.

Motor proteins: myosin V--the multi-purpose transport motor.

Studies in yeast and mice suggest that myosin V participates in the directed transport of a number of distinct cargos to polarized regions of the cell; myosin V has also been implicated in the provision of materials for filopodial extension in neurons.

Getting to the point with myosin VI.

The recent observation that class VI myosins move in a direction opposite to all other known myosins, taken together with analyses of mutant flies and mice, suggests that, instead of moving vesicles out towards the cell periphery, myosin VI more likely brings materials or membranes into the cell.

A class VII unconventional myosin is required for phagocytosis.

BACKGROUND: Phagocytosis, the process by which cells internalize particles, is essential for the defense of multicellular organisms against invading pathogens and is the major means by which many unicellular organisms obtain nutrients. The actin cytoskeleton plays a critical role in phagocytosis and the observation that a significant amount of force (10-20 nN) is generated during internalization, suggests that a myosin participates in the process. Although more than 15 distinct classes of myosin have been identified, their roles in phagocytosis are unknown. RESULTS: The identification of a class VII unconventional myosin (DdMVII) in the Dictyostelium discoideum amoeba, which is a model for phagocytosis, is reported here. Mutant cells lacking DdMVII exhibited an 80% decrease in the uptake of particles whereas all other actin-based behaviors that were tested, including pinocytosis, exocytosis, cytokinesis and morphogenesis, proceeded normally. The defect in phagocytosis was neither because of altered particle binding nor inability to form actin-filled phagocytic cups. CONCLUSIONS: Molecular genetic analysis of Dictyostelium myosin VII reveals that this motor protein plays a specific and significant role in phagocytosis.

Intracellular motility: how can we all work together?

Recent results reinforce the view that actin-based and microtubule-based motility systems do not operate independently, but are used in coordinated fashion to determine intracellular localization of cargo such as organelles.

A role for myosin VII in dynamic cell adhesion.

BACKGROUND: The initial stages of phagocytosis and cell motility resemble each other. The extension of a pseudopod at the leading edge of a migratory cell and the formation of a phagocytic cup are actin dependent, and each rely on the plasma membrane adhering to a surface during dynamic extension. RESULTS: A myosin VII null mutant exhibited a drastic loss of adhesion to particles, consistent with the extent of an observed decrease in particle uptake. Additionally, cell-cell adhesion and the adhesion of the leading edge to the substratum during cell migration were defective in the myosin VII null cells. GFP-myosin VII rescued the phagocytosis defect of the null mutant and was distributed in the cytosol and recruited to the cortical cytoskeleton, where it appeared to be enriched at the tips of filopods. It was also localized to phagocytic cups, but only during the initial stages of particle engulfment. During migration, GFP-myosin VII is found at the leading edge of the cell. CONCLUSIONS: Myosin VII plays an important role in mediating the initial binding of cells to substrata, a novel role for an unconventional myosin.

Myosin VI is required for asymmetric segregation of cellular components during C. elegans spermatogenesis.

BACKGROUND: The asymmetric division of cells and unequal allocation of cell contents is essential for correct development. This process of active segregation is poorly understood but in many instances has been shown to depend on the cytoskeleton. Motor proteins moving along actin filaments and microtubules are logical candidates to provide the motive force for asymmetric sorting of cell contents. The role of myosins in such processes has been suggested, but few examples of their involvement are known. RESULTS: Analysis of a Caenorhabditis elegans class VI myosin deletion mutant reveals a role for this motor protein in the segregation of cell components during spermatogenesis. Mutant spermatocytes cannot efficiently deliver mitochondria and endoplasmic reticulum/Golgi-derived fibrous-body membranous organelle complexes to budding spermatids, and fail to remove actin filaments and microtubules from the spermatids. The segregation defects are not due to a global sorting failure as nuclear inheritance is unaffected. CONCLUSIONS: C. elegans myosin VI has an important role in the unequal partitioning of both organelles and cytoskeletal components, a novel role for this class of motor protein.

The myosin I SH3 domain and TEDS rule phosphorylation site are required for in vivo function.

The class I myosins play important roles in controlling many different types of actin-based cell movements. Dictyostelium cells either lacking or overexpressing amoeboid myosin Is have significant defects in cortical activities such as pseudopod extension, cell migration, and macropinocytosis. The existence of Dictyostelium null mutants with strong phenotypic defects permits complementation analysis as a means of exploring important functional features of the myosin I heavy chain. Mutant Dictyostelium cells lacking two myosin Is exhibit profound defects in growth, endocytosis, and rearrangement of F-actin. Expression of the full-length myoB heavy chain in these cells fully rescues the double mutant defects. However, mutant forms of the myoB heavy chain in which a serine at the consensus phosphorylation site has been altered to an alanine or in which the C-terminal SH3 domain has been removed fail to complement the null phenotype. The wild-type and mutant forms of the myoB heavy chain appeared to be properly localized when they were expressed in the myosin I null mutants. These results suggest that the amoeboid myosin I consensus phosphorylation site and SH3 domains do not play a role in the localization of myosin I, but are absolutely required for in vivo function.

The unconventional myosin encoded by the myoA gene plays a role in Dictyostelium motility.

The myoA gene of Dictyostelium is a member of a gene family of unconventional myosins. The myosin Is share homologous head and basic domains, but the myoA gene product lacks the glycine-, proline-, alanine-rich and src homology 3 domains typical of several of the other myosin Is. A mutant strain of Dictyostelium lacking a functional myoA gene was produced by gene targeting, and the motility of this strain in buffer and a spatial gradient of the chemoattractant cyclic AMP was analyzed by computer-assisted methods. The myoA- cells have a normal elongate morphology in buffer but exhibit a decrease in the instantaneous velocity of cellular translocation, an increase in the frequency of lateral pseudopod formation, and an increase in turning. In a spatial gradient, in which the frequency of pseudopod formation is depressed, myoA- cells exhibit positive chemotaxis but still turn several times more frequently than control cells. These results demonstrate that the other members of the unconventional myosin family do not fully compensate for the loss of functional myoA gene product. Surprisingly, the phenotype of the myoA- strain closely resembles that of the myoB- strain, suggesting that both play a role in the frequency of pseudopod formation and turning during cellular translocation.

A family of unconventional myosins from the nematode Caenorhabditis elegans.

The unconventional myosins are a superfamily of actin-based motor proteins that are expressed in a wide range of cell types and organisms. Thirteen classes of unconventional myosin have been defined, and current efforts are focused on elucidating their individual functions in vivo. Here, we report the identification of a family of unconventional myosin genes in Caenorhabditis elegans. The hum-1, hum-2, hum-3 and hum-6 (heavy chain of an unconventional myosin) genes encode members of myosin classes I, V, VI and VII, respectively. The hum-4 gene encodes a high molecular mass myosin (ca 307 kDa) that is one of the most highly divergent myosins, and is the founding and only known member of class XII. The physical position of each hum gene has been determined. The hum-1, hum-2 and hum-3 genes have been mapped by extrapolation near previously uncharacterized mutations, several of which are lethal, identifying potentially essential unconventional myosin genes in C. elegans.

A potential mechanism for regulating myosin I binding to membranes in vivo.

Myosin Is are associated with specific membranes, however, the mechanism for regulating their intracellular localization is unclear. As a first step towards understanding this mechanism, membrane rebinding assays using Dictyostelium myoB were performed. Crude, cytosolic myoB bound to intact, but not to NaOH-treated plasma membranes. In contrast, partially purified myoB binds to both intact and NaOH-treated plasma membranes. Chemical cross-linking of cytosolic myoB yielded several products, whereas none were found with the partially purified myoB. These results suggest a model where proteins regulating the specific binding of myoB to the plasma membrane may exist both in the cytosol and on the plasma membrane.

Contemporary issues in the psychotherapy of women.

According to current research findings, women are more likely than men to suffer from depression, eating disorders, and multiple personality disorder. The authors review recent advances in the understanding of female psychology, family systems theory, and psychoanalytic theory to show how women's unique developmental challenges and complex social conditions interact to determine the form and expression of these disorders. After discussing contributing factors, the authors suggest appropriate approaches to diagnosis and treatment. They conclude that therapists of both genders must appreciate the complex causes of these disorders to be most helpful to their female patients.