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).

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.

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.

Recruitment of a specific amoeboid myosin I isoform to the plasma membrane in chemotactic Dictyostelium cells.

The Dictyostelium class I myosins, MyoA, -B, -C, and -D, participate in plasma membrane-based cellular processes such as pseudopod extension and macropinocytosis. Given the existence of a high affinity membrane-binding site in the C-terminal tail domain of these motor proteins and their localized site of action at the cortical membrane-cytoskeleton, it was of interest to determine whether each myosin I was directly associated with the plasma membrane. The membrane association of a myosin I heavy chain kinase that regulates the activity of one of the class I myosins, MyoD was also examined. Cellular fractionation experiments revealed that the majority of the Dicyostelium MyoA, -B, -C and -D heavy chains and the kinase are cytosolic. However, a small, but significant, fraction (appr. 7. -15%) of each myosin I and the kinase was associated with the plasma membrane. The level of plasma membrane-associated MyoB, but neither that of MyoC nor MyoD, increases up to 2-fold in highly motile, streaming cells. These results indicate that Dictyostelium specifically recruits myoB to the plasma membrane during directed cell migration, consistent with its known role in pseudopod formation.

Myosin I phosphorylation is increased by chemotactic stimulation.

Directed cell migration occurs in response to extracellular cues. Following stimulation of a cell with chemoattractant, a significant rearrangement of the actin cytoskeleton is mediated by intracellular signaling pathways and results in polarization of the cell and movement via pseudopod extension. Amoeboid myosin Is play a critical role in regulating pseudopod formation in Dictyostelium, and their activity is activated by heavy chain phosphorylation. The effect of chemotactic stimulation on the in vivo phosphorylation level of a Dictyostelium myosin I, myoB, was tested. The myoB heavy chain is phosphorylated in vivo on serine 322 (the myosin TEDS rule phosphorylation site) in chemotactically competent cells. The level of myoB phosphorylation increases following stimulation of starving cells with the chemoattractant cAMP. A 3-fold peak increase in the level of phosphorylation is observed at 60 s following stimulation, a time at which the Dictyostelium cell actively extends pseudopodia. These findings suggest that chemotactic stimulation results in increased myoB activity via heavy chain phosphorylation and contributes to the global extension of pseudopodia that occurs prior to polarization and directed motility.

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.

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.

The role of unconventional myosins in Dictyostelium endocytosis.

Dictyostelium discoideum is a simple eukaryote amenable to detailed molecular studies of the endocytic processes phagocytosis and macropinocytosis. Both the actin cytoskeleton and associated myosin motors are well-described and a range of mutants are now available that enable characterization of the role of the cytoskeleton in a range of cellular functions. Molecular genetic studies have uncovered roles for two different classes of Dictyostelium unconventional myosins in endocytosis. The class I myosins contribute to both macropinocytosis and phagocytosis by playing a general role in controlling actin-dependent manipulations of the actin-rich cortex. A class VII myosin has been shown to be important for phagocytosis. This brief review summarizes what is known about the role of these different myosins in both fluid and particle uptake in this system.

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.

Unconventional myosins: anchors in the membrane traffic relay.

The family of unconventional myosins is ever growing and the functions attributed to them seem to expand in parallel. These actin-based motor proteins have been implicated in processes as seemingly diverse as endocytosis and exocytosis, the transport of organelles, in spermatogenesis and in neurosensory functions such as hearing and sight. A common myosin function may underlie them all--the regulation of intracellular membrane traffic.

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.

Myosin I contributes to the generation of resting cortical tension.

The amoeboid myosin I's are required for cellular cortical functions such as pseudopod formation and macropinocytosis, as demonstrated by the finding that Dictyostelium cells overexpressing or lacking one or more of these actin-based motors are defective in these processes. Defects in these processes are concomitant with changes in the actin-filled cortex of various Dictyostelium myosin I mutants. Given that the amoeboid myosin I's possess both actin- and membrane-binding domains, the mutant phenotypes could be due to alterations in the generation and/or regulation of cell cortical tension. This has been directly tested by analyzing mutant Dictyostelium that either lacks or overexpresses various myosin I's, using micropipette aspiration techniques. Dictyostelium cells lacking only one myosin I have normal levels of cortical tension. However, myosin I double mutants have significantly reduced (50%) cortical tension, and those that mildly overexpress an amoeboid myosin I exhibit increased cortical tension. Treatment of either type of mutant with the lectin concanavalin A (ConA) that cross-links surface receptors results in significant increases in cortical tension, suggesting that the contractile activity of these myosin I's is not controlled by this stimulus. These results demonstrate that myosin I's work cooperatively to contribute substantially to the generation of resting cortical tension that is required for efficient cell migration and macropinocytosis.

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.

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.

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.

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.

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.

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.