Kathryn G. Miller

A role for myosin VI in actin dynamics at sites of membrane remodeling during Drosophila spermatogenesis

Myosin VI has been implicated in membrane dynamics in several organisms. The mechanism of its participation in membrane events is not clear. We have used spermatogenesis in Drosophila to investigate myosin VIs in vivo role. We demonstrate that myosin VI colocalizes with and is required for the accumulation of the actin polymerization regulatory proteins, cortactin and arp2/3 complex, on actin structures that mediate membrane remodeling during spermatogenesis. In addition, we show that dynamin localizes to these actin structures and that when dynamin and myosin VI function are both impaired, major defects in actin structures are observed. We conclude that during spermatogenesis myosin VI and dynamin function in parallel pathways that regulate actin dynamics and that cortactin and arp2/3 complex may be important for these functions. Regions of myosin VI accumulation are proposed as sites where actin assembly is coupled to membrane dynamics.

The actin cytoskeleton is required for maintenance of posterior pole plasm components in the Drosophila embryo.

Localization of mRNAs is one of many aspects of cellular organization that requires the cytoskeleton. In Drosophila, microtubules are known to be required for correct localization of developmentally important mRNAs and proteins during oogenesis; however, the role of the actin cytoskeleton in localization is less clear. Furthermore, it is not known whether either of these cytoskeletal systems are necessary for maintenance of RNA localization in the early embryo. We have examined the contribution of the actin and microtubule cytoskeletons to maintenance of RNA and protein localization in the early Drosophila embryo. We have found that while microtubules are not necessary, the actin cytoskeleton is needed for stable association of nanos, oskar, germ cell-less and cyclin B mRNAs and Oskar and Vasa proteins at the posterior pole in the early embryo. In contrast, bicoid RNA, which is located at the anterior pole, does not require either cytoskeletal system to remain at the anterior.

Use of actin filament and microtubule affinity chromatography to identify proteins that bind to the cytoskeleton

Publisher Summary The proteins that bind to actin filaments and microtubules play an important role in determining the structure and function of these filaments in eukaryotic cells. A number of approaches have been used to identify and characterize these proteins. Actin-binding proteins (ABPs) have been identified primarily by virtue of their ability to affect actin polymerization or actin filament motility in vitro . This chapter describes affinity chromatography methods for the isolation of proteins that bind to actin filaments and microtubules. The use of affinity chromatography offers a number of advantages over the use of other procedures for the purification of cytoskeletal proteins. Large extents of purification are obtained without requiring an activity assay. The agarose matrix used for construction of affinity columns consists of a 1:1 mixture of Affi-Gel 10 and Sepharose CL-6B. Affinity columns constructed from stabilized actin filaments and microtubules will contain a large proportion of protein that is not covalently bound to the column matrix, since a filament needs to be linked to the matrix only at scattered sites to remain bound to the column.

Proper Cellular Reorganization during Drosophila spermatid Individualization Depends on Actin Structures Composed of Two Domains, Bundles and Meshwork, that Are Differentially Regulated and Have Different Functions

During spermatid individualization in Drosophila, actin structures (cones) mediate cellular remodeling that separates the syncytial spermatids into individual cells. These actin cones are composed of two structural domains, a front meshwork and a rear region of parallel bundles. We show here that the two domains form separately in time, are regulated by different sets of actin-associated proteins, can be formed independently, and have different roles. Newly forming cones were composed only of bundles, whereas the meshwork formed later, coincident with the onset of cone movement. Polarized distributions of myosin VI, Arp2/3 complex, and the actin-bundling proteins, singed (fascin) and quail (villin), occurred when movement initiated. When the Arp2/3 complex was absent, meshwork formation was compromised, but surprisingly, the cones still moved. Despite the fact that the cones moved, membrane reorganization and cytoplasmic exclusion were abnormal and individualization failed. In contrast, when profilin, a regulator of actin assembly, was absent, bundle formation was greatly reduced. The meshwork still formed, but no movement occurred. Analysis of this actin structures formation and participation in cellular reorganization provides insight into how the mechanisms used in cell motility are modified to mediate motile processes within specialized cells.

Coiled-Coil–Mediated Dimerization Is Not Required for Myosin VI to Stabilize Actin during Spermatid Individualization in Drosophila melanogaster

Myosin VI is a pointed-end-directed actin motor that is thought to function as both a transporter of cargoes and an anchor, capable of binding cellular components to actin for long periods. Dimerization via a predicted coiled coil was hypothesized to regulate activity and motor properties. However, the importance of the coiled-coil sequence has not been tested in vivo. We used myosin VIs well-defined role in actin stabilization during Drosophila spermatid individualization to test the importance in vivo of the predicted coiled coil. If myosin VI functions as a dimer, a forced dimer should fully rescue myosin VI loss of function defects, including actin stabilization, actin cone movement, and cytoplasmic exclusion by the cones. Conversely, a molecule lacking the coiled coil should not rescue at all. Surprisingly, neither prediction was correct, because each rescued partially and the molecule lacking the coiled coil functioned better than the forced dimer. In extracts, no cross-linking into higher molecular weight forms indicative of dimerization was observed. In addition, a sequence required for altering nucleotide kinetics to make myosin VI dimers processive is not required for myosin VIs actin stabilization function. We conclude that myosin VI does not need to dimerize via the predicted coiled coil to stabilize actin in vivo.

A role for moesin in polarity.

Three groups have recently characterized defects arising in development owing to mutations in the gene encoding Dmoesin, which is the sole ezrin-radixin-moesin (ERM) protein in Drosophila. Previously, studies in cultured mammalian cells suggested that ERM proteins are important for actin-membrane associations. However, mutations in moesin and radixin in mice do not cause severe defects, indicating functional overlap among vertebrate ERM paralogs. In Drosophila, however, mutations in Dmoesin result in lethality. Actin organization in imaginal disc epithelia is abnormal and apical-basal polarity is lost. When moesin function is reduced in the female germ-line, defects in cortical actin organization are also observed. Localization of informational molecules at the oocyte posterior is strongly affected, thus indicating a role for moesin in anchoring these determinants.

Androcam Is a Tissue-specific Light Chain for Myosin VI in the Drosophila Testis

Myosin VI, a ubiquitously expressed unconventional myosin, has roles in a broad array of biological processes. Unusual for this motor family, myosin VI moves toward the minus (pointed) end of actin filaments. Myosin VI has two light chain binding sites that can both bind calmodulin (CaM). However unconventional myosins could use tissue-specific light chains to modify their activity. In the Drosophila testis, myosin VI is important for maintenance of moving actin structures, called actin cones, which mediate spermatid individualization. A CaM-related protein, Androcam (Acam), is abundantly expressed in the testis and like myosin VI, accumulates on these cones. We have investigated the possibility that Acam is a testis-specific light chain of Drosophila myosin VI. We find that Acam and myosin VI precisely colocalize at the leading edge of the actin cones and that myosin VI is necessary for this Acam localization. Further, myosin VI and Acam co-immunoprecipitate from the testis and interact in yeast two-hybrid assays. Finally Acam binds with high affinity to peptide versions of both myosin VI light chain binding sites. In contrast, although Drosophila CaM also shows high affinity interactions with these peptides, we cannot detect a CaM/myosin VI interaction in the testis. We conclude that Acam and not CaM acts as a myosin VI light chain in the Drosophila testis and hypothesize that it may alter the regulation of myosin VI in this tissue.

Evolutionary implications of developmental instability in parthenogenetic Drosophila mercatorum. I. Comparison of several strains with different genotypes

SUMMARY Natural populations of sexually reproducing Drosophila mercatorum are capable of a very low rate of parthenogenesis, but this mode of reproduction has apparently never characterized an entirely asexual population in this species. The high abortion rate observed in laboratory parthenogenetic lines suggests that developmental constraints may cause the failure of this trait to spread in nature. To investigate the basis of this developmental instability and how it may affect the evolution of parthenogenesis in natural populations, early embryonic development was compared between one sexual and four parthenogenetic laboratory strains of D. mercatorum. There is a large amount of variation within a given parthenogenetic strain, suggesting that parthenogenesis is associated with a general breakdown of developmental stability. There is relatively little variation among different parthenogenetic strains, suggesting that most abortions are due to a feature inherent to parthenogenetic reproduction rather than a feature of a particular genome. Likewise, there is little variation between parthenogenetic and sexual strains in the causes of abortions, suggesting that the developmental problems encountered by parthenogenetic lineages are not unique to parthenogens. Thus, the failure of parthenogenesis to spread within D. mercatorum can be attributed to no particular developmental constraint per se operating after the initiation of embryogenesis. However, the overall increase in all developmental problems that occurs with the transition from sexual to parthenogenetic development suggests that the high degree of developmental instability associated with parthenogenesis may be considered a developmental constraint in its own right.

Extending the Arp2/3 complex and its regulation beyond the leading edge

Two studies characterizing Drosophila Arp2/3 complex and Scar mutants demonstrate that assembly of some actin structures in nonmotile cells of multicellular organisms utilizes the same proteins as are important for actin assembly in motile cells. These studies also show that assembly of other actin structures is independent of these proteins, suggesting that alternative mechanisms also exist.

PULSE Vision & Change Rubrics

Dear Editor: We want to inform CBE—Life Sciences Education readers about the release of the first version of the Partnership for Undergraduate Life Sciences Education (PULSE) Vision & Change Rubrics (available at the PULSE Community website: www.pulsecommunity.org). These rubrics were written and assembled by the PULSE Vision & Change Leadership Fellows to help stimulate the widespread adoption of the principles outlined in the 2011 National Academy of Sciences report Vision and Change in Undergraduate Biology Education: A Call to Action (American Association for the Advancement of Science [AAAS], 2011 ). Initially, these rubrics can be utilized for departmental self-assessment. In the longer term, the rubrics are intended serve as the basis of a tiered certification program for life sciences departments based on Vision and Change principles. In 2006, the National Science Foundation (NSF) initiated a multi-year conversation with the undergraduate life sciences community, with assistance from the AAAS. That conversation, cosponsored by the National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) and the Howard Hughes Medical Institute (HHMI), resulted in the release of the Vision and Change report in 2011 (AAAS, 2011 ). Among the recommendations in the Vision and Change report was recognition that a 21st-century education requires modifications of faculty incentive systems, academic departmental support, how curricular decisions are determined, and biology education pedagogy. In 2012, the NSF, NIH/NIGMS, and HHMI founded the Partnership for Undergraduate Life Sciences Education, or PULSE, to catalyze implementation of Vision and Change principles across all institutions of higher education. The PULSE community, open to all life sciences educators, now hosts more than 1000 members. Forty Vision and Change Leadership Fellows selected from among biologists with leadership roles at institutions of higher education of all types were charged with developing strategies to promote systemic changes in life sciences education. The PULSE initiative is intended to catalyze change at the departmental and institutional levels by initiating and implementing new strategies to assist departments and institutions to move toward a shared vision and effect curricular transformation (Manning, 2013 ). Decisions regarding curriculum, hiring, teaching assignments, faculty evaluations, mentoring, and faculty development are typically made at the department level. Thus, a concerted effort at this level is needed to overcome the widespread resistance to change (Savkar and Lokere, 2010 ; Anderson et al., 2011 ). During the past year, the 40 PULSE Leadership Fellows have designed and begun to implement several initiatives to facilitate educational transformation in life sciences departments. One of these initiatives, the PULSE Vision & Change Rubrics, is described briefly here. The PULSE Vision & Change Rubrics articulate fundamental criteria for evaluating the level and degree of departmental adoption of the principles of Vision and Change. These rubrics assess department or program alignment with Vision and Change recommendations in five broad areas: curriculum alignment, assessment, faculty practice/faculty support, infrastructure, and climate for change. Each rubric has several categories with multiple criteria to be assessed (see Figure 1 for a sample rubric). The rubric descriptors designate different levels of implementation of Vision and Change principles from first steps to full departmental transformation. The set of rubrics has been designed for flexible use by undergraduate life sciences departments at a broad range of institution types including 2-yr colleges, 4-yr liberal arts institutions, regional comprehensive institutions, and research institutions. Figure 1. Sample from the PULSE Vision & Change Rubrics. The rubrics are organized with separate criteria listed on the left, with levels of achievement across the top, from zero (no achievement) to four (exemplary achievement). In total, 69 separate criteria ... We expect that the PULSE Vision & Change Rubrics will be used for a variety of purposes, including departmental self-assessment, engagement of senior academic administrators, and as the basis for a departmental certification program. Initially, the rubrics can provide a structure and “road map” for departmental reflection regarding a host of topics relevant to implementation of Vision and Change recommendations. A goal of the rubrics is to provide a basic framework of expectations, such that evidence of adoption of Vision and Change principles can be gathered and self-assessed by departments and a road map for continued transformation can be charted. We anticipate that the rubrics themselves can serve as an assessment tool to examine how changes in departmental practices over time affect student outcomes. Longer term, we intend the rubrics to serve as the basis for a tiered certification program for undergraduate life sciences departments that have adopted some or all of the principles outlined in the Vision and Change report. Certification will serve both to reward departments that have made substantial progress in implementing Vision and Change, and to incentivize departments that have been slow to adopt these changes. An underlying assumption of the PULSE Vision & Change Rubrics is that higher scores resulting from excellent/exemplar level of achievement will result in better student outcomes. At present, this is only a hypothesis, and we and others who use the rubrics will gather data to test this hypothesis. This evidence will guide future revisions of the PULSE Vision & Change Rubrics, to ensure that they reflect the criteria that “really matter” to improve student learning. We are very interested in receiving feedback from the biology education community. This is most easily done via the PULSE Community website or by contacting one of the authors directly. During the past year of development, we have done our best to include the most relevant criteria for capturing and recognizing departmental efforts, but it is likely we have omitted key items or areas. We are depending on the community to provide feedback so as to make the rubrics as effective as possible. We anticipate that the rubrics will be revised over the next year, and we expect them to evolve in future years based on evidence of their effectiveness and changing standards of knowledge and practice in undergraduate life sciences education.