Search

A useful model for determining the mechanisms by which actin and actin binding proteins control cellular architecture is the

Background Local and national initiatives to improve the learning experiences of students enrolled in Science, Technology, Engineering, and Mathematics (STEM) courses have been on-going for a couple of decades with a heightened momentum within the last 10 years. However, recent large-scale studies have demonstrated that transmission of information is still the primary mode of instruction in STEM courses across the undergraduate curriculum. The limited impact of instructional change reform efforts can be partly explained by the one-sided focus of educational research on the development of evidence-based instructional practices and production of evidence demonstrating their impact on student learning. This has been done at the expense of understanding faculty members’ instructional practices and mindsets about teaching and learning that underlie their practices. This study addresses this gap in the literature by characterizing STEM instructors’ instructional intentions and reflections on their teaching performance for a week of instruction. Data was collected through semi-structured interviews with 42 STEM faculty members from one doctorate-granting institution in the USA. Results STEM instructors in this study had teacher-centric mindsets with respect to their instructional planning (e.g., content-focused learning goals, lecture is seen as an engagement strategy). We found that these instructors mostly saw formative assessment tools as engagement strategy rather than tools to monitor student learning. Reflections on their level of satisfaction with their week of teaching focused heavily on content coverage and personal feelings and minimally considered student learning. Finally, we found that pedagogical discontent was not a driver of planned course revisions. Conclusions This study identifies mismatches between STEM instructors’ teaching mindsets and current approaches to instructional change. STEM instructors in this study paid minimal attention to student learning when considering course-level revisions and many of their reflections were anchored in their personal feelings. However, instructional reform strategies often attempt to convince faculty of a new approach by demonstrating its impact on student learning. The misalignment identified in this study further highlights the need to better characterize STEM instructors’ cognition around teaching so that reform efforts can better meet them where they are.

Objective: Rett syndrome (RTT) and epileptic encephalopathy (EE) are devastating neurodevelopmental disorders with distinct diagnostic criteria. However, highly heterogeneous and overlapping clinical features often allocate patients into the boundary of the two conditions, complicating accurate diagnosis and appropriate medical interventions. Therefore, we investigated the specific molecular mechanism that allows an understanding of the pathogenesis and relationship of these two conditions. Methods: We screened novel genetic factors from 34 RTT-like patients without MECP2 mutations, which account for ∼90% of RTT cases, by whole exome sequencing. The biological function of the discovered variants was assessed in cell culture and Xenopus tropicalis models. Results: We identified a recurring de novo variant in GABAB receptor R2 (GABBR2) that reduces the receptor function, whereas different GABBR2 variants in EE patients possess a more profound effect in reducing receptor activity and are more responsive to agonist rescue in an animal model. Interpretation: GABBR2 is a genetic factor that determines RTT- or EE-like phenotype expression depending on the variant positions. GABBR2-mediated GABA signaling is a crucial factor in determining the severity and nature of neurodevelopmental phenotypes. This article is protected by copyright. All rights reserved.

Myosin VI (MVI) is a versatile actin-based motor protein that has been implicated in a variety of different cellular processes, including endo- and exocytic vesicle trafficking, Golgi morphology, and actin structure stabilization. A role for MVI in crucial actin-based processes involved in sperm maturation was demonstrated in Drosophila. Because of the prominence and importance of actin structures in mammalian spermiogenesis, we investigated whether MVI was associated with actin-mediated maturation events in mammals. Both immunofluorescence and ultrastructural analyses using immunogold labeling showed that MVI was strongly linked with key structures involved in sperm development and maturation. During the early stage of spermiogenesis, MVI is associated with the Golgi and with coated and uncoated vesicles, which fuse to form the acrosome. Later, as the acrosome spreads to form a cap covering the sperm nucleus, MVI is localized to the acroplaxome, an actin-rich structure that anchors the acrosome to the nucleus. Finally, during the elongation/maturation phase, MVI is associated with the actin-rich structures involved in nuclear shaping: the acroplaxome, manchette, and Sertoli cell actin hoops. Since this is the first report of MVI expression and localization during mouse spermiogenesis and MVI partners in developing sperm have not yet been identified, we discuss some probable roles for MVI in this process. During early stages, MVI is hypothesized to play a role in Golgi morphology and function as well as in actin dynamics regulation important for attachment of developing acrosome to the nuclear envelope. Next, the protein might also play anchoring roles to help generate forces needed for spermatid head elongation. Moreover, association of MVI with actin that accumulates in the Sertoli cell ectoplasmic specialization and other actin structures in surrounding cells suggests additional MVI functions in spermatid movement across the seminiferous epithelium and in sperm release.

Variants in IQSEC2, escaping X inactivation, cause X-linked intellectual disability with frequent epilepsy in males and females. We aimed to investigate sex-specific differences., SCOPUS: ar.j, info:eu-repo/semantics/published

This Article was originally published under Nature Research’s License to Publish, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the Article have been modified accordingly.

Purpose: To define the clinical characteristics of patients with variants in TCF20, we describe 27 patients, 26 of whom were identified via exome sequencing. We compare detailed clinical data with 17 previously reported patients. Methods: Patients were ascertained through molecular testing laboratories performing exome sequencing (and other testing) with orthogonal confirmation; collaborating referring clinicians provided detailed clinical information. Results: The cohort of 27 patients all had novel variants, and ranged in age from two to 68 years. All had developmental delay/intellectual disability. Autism spectrum disorders/autistic features were reported in 69%, attention disorders or hyperactivity in 67%, craniofacial features (no recognizable facial gestalt) in 67%, structural brain anomalies in 24%, and seizures in 12%. Additional features affecting various organ systems were described in 93%. In a majority of patients, we did not observe previously reported findings of postnatal overgrowth or craniosynostosis, in comparison to earlier reports. Conclusion: We provide valuable data regarding the prognosis and clinical manifestations of patients with variants in TCF20.

Tatton-Brown-Rahman syndrome (TBRS; OMIM 615879), also known as the DNMT3A-overgrowth syndrome, is an overgrowth intellectual disability syndrome first described in 2014 with a report of 13 individuals with constitutive heterozygous DNMT3A variants. Here we have undertaken a detailed clinical study of 55 individuals with de novoDNMT3A variants, including the 13 previously reported individuals. An intellectual disability and overgrowth were reported in >80% of individuals with TBRS and were designated major clinical associations. Additional frequent clinical associations (reported in 20-80% individuals) included an evolving facial appearance with low-set, heavy, horizontal eyebrows and prominent upper central incisors; joint hypermobility (74%); obesity (weight ³2SD, 67%); hypotonia (54%); behavioural/psychiatric issues (most frequently autistic spectrum disorder, 51%); kyphoscoliosis (33%) and afebrile seizures (22%). One individual was diagnosed with acute myeloid leukaemia in teenage years. Based upon the results from this study, we present our current management for individuals with TBRS. ispartof: Wellcome Open Res vol:3 pages:46- ispartof: location:England status: Published online

Disruption of the establishment of left-right (L-R) asymmetry leads to situs anomalies ranging from situs inversus totalis (SIT) to situs ambiguus (heterotaxy). The genetic causes of laterality defects in humans are highly heterogeneous. Via whole-exome sequencing (WES), we identified homozygous mutations in PKD1L1 from three affected individuals in two unrelated families. PKD1L1 encodes a polycystin-1-like protein and its loss of function is known to cause laterality defects in mouse and medaka fish models. Family 1 had one fetus and one deceased child with heterotaxy and complex congenital heart malformations. WES identified a homozygous splicing mutation, c.6473+2_6473+3delTG, which disrupts the invariant splice donor site in intron 42, in both affected individuals. In the second family, a homozygous c.5072G>C (p.Cys1691Ser) missense mutation was detected in an individual with SIT and congenital heart disease. The p.Cys1691Ser substitution affects a highly conserved cysteine residue and is predicted by molecular modeling to disrupt a disulfide bridge essential for the proper folding of the G protein-coupled receptor proteolytic site (GPS) motif. Damaging effects associated with substitutions of this conserved cysteine residue in the GPS motif have also been reported in other genes, namely GPR56 , BAI3 , and PKD1 in human and lat-1 in C. elegans , further supporting the likely pathogenicity of p.Cys1691Ser in PKD1L1. The identification of bi-allelic PKD1L1 mutations recapitulates previous findings regarding phenotypic consequences of loss of function of the orthologous genes in mice and medaka fish and further expands our understanding of genetic contributions to laterality defects in humans.

Morbidity and Mortality conference (MMC) serves an important role in medical care and education. We restructured our Department of Pediatrics MMC to focus on multidisciplinary participation and improved communication among disciplines, quality improvement, and system changes for safer clinical care and enhanced learning from adverse outcomes.The structure and philosophy of the Department of Pediatrics MMC was changed. We present guiding principles for the restructuring process and evaluation results postrestructuring, which examined achievement of conference goals, including quality improvement.The MMC led to system changes within the Department of Pediatrics as well as other parts of the hospital. Satisfaction with these changes was high among conference participants, who felt that the conference achieved its goals of including multiple disciplines and creating system changes.The successful change in the focus of the pediatric MMC conference resulted in significant hospital-wide system changes, quality improvements, enhanced education, and departmental satisfaction.

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 VI's 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 VI's actin stabilization function. We conclude that myosin VI does not need to dimerize via the predicted coiled coil to stabilize actin in vivo.

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 structure's 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.

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.

Profilin is a well-characterized protein known to be important for regulating actin filament assembly. Relatively few studies have addressed how profilin interacts with other actin-binding proteins in vivo to regulate assembly of complex actin structures. To investigate the function of profilin in the context of a differentiating cell, we have studied an instructive genetic interaction between mutations in profilin (chickadee) and capping protein (cpb). Capping protein is the principal protein in cells that caps actin filament barbed ends. When its function is reduced in the Drosophila bristle, F-actin levels increase and the actin cytoskeleton becomes disorganized, causing abnormal bristle morphology. chickadee mutations suppress the abnormal bristle phenotype and associated abnormalities of the actin cytoskeleton seen in cpb mutants. Furthermore, overexpression of profilin in the bristle mimics many features of thecpb loss-of-function phenotype. The interaction betweencpb and chickadee suggests that profilin promotes actin assembly in the bristle and that a balance between capping protein and profilin activities is important for the proper regulation of F-actin levels. Furthermore, this balance of activities affects the association of actin structures with the membrane, suggesting a link between actin filament dynamics and localization of actin structures within the cell.

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 VI's 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.

Actin is a highly conserved protein found in all eukaryotic organisms. Most organisms have multiple cytoplasmic actin genes that encode isoforms with slightly different amino acid sequences. These different isoforms are coexpressed in many cell types. Why organisms have multiple very similar cytoplasmic actin genes is unclear. We have addressed this question with the cytoplasmic actins in Drosophila , Act5C, and Act42A. These isoforms differ by only two amino acids and both genes are expressed in all cells at all times during development. We identified P element insertions in the Act5C gene that resulted in a lethal phenotype. The lethal phenotype is rescued by a transgene with a genomic fragment that includes Act5C regulatory and amino acid coding sequences. A hybrid transgene containing the protein coding sequence for the Act42A isoform, under the control of the regulatory regions of the Act5C gene, also rescues the lethality of the Act5C mutants. Furthermore, flies that carry only one copy each of Act5C and Act42A are viable. These results suggest the amino acid differences between these two cytoplasmic actin isoforms are not important for function and the need for increased gene dosage to provide more actin is not likely to explain the existence of multiple genes. Instead, our results suggest that regulated expression of Act5C is essential to the fly.

Developmental instability is particularly pronounced in parthenogenetic strains of Drosophila mercatorum. All parthenogenetically produced eggs in a given strain have the same genotype, but even when reared in the same environment, only approximately 5% of the eggs initiating development ever reach adulthood. A sexual analogue of a parthenogenetic strain was created to investigate the basis of this developmental instability. The two strains have identical genotypes (except for the Y chromosome in males of the sexual strain) and differ only in mode of reproduction. The sexual strain had a much lower rate of developmental instability than the parthenogenetic strain, suggesting that the instability is caused by the mode of reproduction per se and is not due to homozygosity, disruption of coadapted gene complexes, or any other feature of the parthenogenetic genome. The increased rate of abortion with parthenogenetic reproduction is caused by a proportional increase in the normal panoply of errors that occur in sexual reproduction but at a much lower rate. Attempts to establish other sexual analogues of laboratory parthenogenetic strains revealed different male sterility factors within several parthenogenetic genomes that could potentially act to prevent hybridization with sexually reproducing ancestors during the incipient stages in the evolution of an entirely parthenogenetic lineage.

Biology education research (BER) can be a major contributor to the herculean task of modernizing and transforming biology education. However, as researchers in a relatively young and still small field, BER practitioners now find themselves fragmented across 64 biology-related societies and lacking agreement on a core research agenda, a convenient professional network, and venues of dissemination. To begin addressing these needs, the newly formed Society for the Advancement of Biology Education Research (SABER) held an inaugural meeting in September 2010 at the University of Minnesota–Twin Cities. The 29 participants (Figure 1) included faculty, postdoctoral fellows, and graduate students engaged in empirical research and professional development, as well as journal editors, textbook writers, and a textbook editor. The diversity of this group contributed to a thoughtful, reflective, and productive meeting, whose major goals were to 1) define BER, 2) identify challenges to its practice, 3) formulate overarching research questions, and 4) outline the role of SABER in supporting the BER community. The consensus views of the participants on each of these goals are described in the paragraphs below. Figure 1: SABER Founding Members: Teri Balser, Clarissa Dirks, Mary Pat Wenderoth, Janet Branchaw, Rob Brooker, Peggy Brickman, Malcolm Campbell, Mark Connelly, Erin Dolan, Scott Freeman, Mark Hens, Jenny Knight, Kathryn Miller, Jennifer Momsen, Lisa Montplaisir, ... The outcomes of this meeting are timely in the context of the recently released summary report Vision and Change in Undergraduate Education, A Call to Action, which charged the biology community with “creating, using, assessing, and disseminating effective practices in teaching and learning” (American Association for the Advancement of Science, 2010 ). By providing the infrastructure needed to create a vibrant network of practitioners, SABER is well positioned to support biology education researchers in generating empirical evidence that can effect meaningful changes in undergraduate biology education.

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.

Coordination of cellular organization requires the interaction of the cytoskeletal filament systems. Recently, several lines of investigation have suggested that transport of cellular components along both microtubules and actin filaments is important for cellular organization and function. We report here on molecules that may mediate coordination between the actin and microtubule cytoskeletons. We have identified a 195-kD protein that coimmunoprecipitates with a class VI myosin, Drosophila 95F unconventional myosin. Cloning and sequencing of the gene encoding the 195-kD protein reveals that it is the first homologue identified of cytoplasmic linker protein (CLIP)–170, a protein that links endocytic vesicles to microtubules. We have named this protein D-CLIP-190 (the predicted molecular mass is 189 kD) based on its similarity to CLIP-170 and its ability to cosediment with microtubules. The similarity between D-CLIP-190 and CLIP-170 extends throughout the length of the proteins, and they have a number of predicted sequence and structural features in common. 95F myosin and D-CLIP-190 are coexpressed in a number of tissues during embryogenesis in Drosophila. In the axonal processes of neurons, they are colocalized in the same particulate structures, which resemble vesicles. They are also colocalized at the posterior pole of the early embryo, and this localization is dependent on the actin cytoskeleton. The association of a myosin and a homologue of a microtubule-binding protein in the nervous system and at the posterior pole, where both microtubule and actin-dependent processes are known to be important, leads us to speculate that these two proteins may functionally link the actin and microtubule cytoskeletons.

Regulation of actin filament length and orientation is important in many actin-based cellular processes. This regulation is postulated to occur through the action of actin-binding proteins. Many actin-binding proteins that modify actin in vitro have been identified, but in many cases, it is not known if this activity is physiologically relevant. Capping protein (CP) is an actin-binding protein that has been demonstrated to control filament length in vitro by binding to the barbed ends and preventing the addition or loss of actin monomers. To examine the in vivo role of CP, we have performed a molecular and genetic characterization of the beta subunit of capping protein from Drosophila melanogaster. We have identified mutations in the Drosophila beta subunit-these are the first CP mutations in a multicellular organism, and unlike CP mutations in yeast, they are lethal, causing death during the early larval stage. Adult files that are heterozygous for a pair of weak alleles have a defect in bristle morphology that is correlated to disorganized actin bundles in developing bristles. Our data demonstrate that CP has an essential function during development, and further suggest that CP is required to regulate actin assembly during the development of specialized structures that depend on actin for their morphology.

This paper describes the development and validation of the PULSE Vision & Change Rubrics, version 1.0, a reliable tool for measuring departmental change. It was found that liberal arts institutions are farther along in implementing the recommendations of Vision and Change and that institutions overall earned the highest scores on the Curriculum rubric and the lowest scores on the Assessment rubric., The PULSE Vision & Change Rubrics, version 1.0, assess life sciences departments’ progress toward implementation of the principles of the Vision and Change report. This paper reports on the development of the rubrics, their validation, and their reliability in measuring departmental change aligned with the Vision and Change recommendations. The rubrics assess 66 different criteria across five areas: Curriculum Alignment, Assessment, Faculty Practice/Faculty Support, Infrastructure, and Climate for Change. The results from this work demonstrate the rubrics can be used to evaluate departmental transformation equitably across institution types and represent baseline data about the adoption of the Vision and Change recommendations by life sciences programs across the United States. While all institution types have made progress, liberal arts institutions are farther along in implementing these recommendations. Generally, institutions earned the highest scores on the Curriculum Alignment rubric and the lowest scores on the Assessment rubric. The results of this study clearly indicate that the Vision & Change Rubrics, version 1.0, are valid and equitable and can track long-term progress of the transformation of life sciences departments. In addition, four of the five rubrics have broad applicability and can be used to evaluate departmental transformation by other science, technology, engineering, and mathematics disciplines.

The Arp2/3 complex and its activators, Scar/WAVE and Wiskott-Aldrich Syndrome protein (WASp), promote actin polymerization in vitro and have been proposed to influence cell shape and motility in vivo. We demonstrate that the Drosophila Scar homologue, SCAR, localizes to actin-rich structures and is required for normal cell morphology in multiple cell types throughout development. In particular, SCAR function is essential for cytoplasmic organization in the blastoderm, axon development in the central nervous system, egg chamber structure during oogenesis, and adult eye morphology. Highly similar developmental requirements are found for subunits of the Arp2/3 complex. In the blastoderm, SCAR and Arp2/3 mutations result in a reduction in the amount of cortical filamentous actin and the disruption of dynamically regulated actin structures. Remarkably, the single Drosophila WASp homologue, Wasp, is largely dispensable for these numerous Arp2/3-dependent functions, whereas SCAR does not contribute to cell fate decisions in which Wasp and Arp2/3 play an essential role. These results identify SCAR as a major component of Arp2/3-dependent cell morphology during Drosophila development and demonstrate that the Arp2/3 complex can govern distinct cell biological events in response to SCAR and Wasp regulation.

Stable actin structures play important roles in the development and specialization of differentiated cells. How these structures form, are organized, and are used to mediate physiological processes is not well understood in most cases. In Drosophila testis, stable actin structures, called actin cones, mediate spermatid individualization, a large-scale cellular remodeling process. These actin cones are composed of two structural domains, a front meshwork and a rear region of parallel bundles. Myosin VI is an important player in proper actin cone organization and function. Myosin VI localizes to the cones' fronts and its specific localization is required for proper actin cone formation and function during individualization. To understand how these structures are organized and assembled, ultrastructural studies are important to reveal both organization of actin and the precise localization of actin regulators relative to regions with different filament organizations. In the present work, we have developed a novel pre-embedding immunogold-silver labeling method for high-resolution analysis of protein distribution in actin structures which allowed both satisfactory antibody labeling and good ultrastructural preservation. Electron microscopic studies revealed that myosin VI accumulated at the extreme leading edge of the actin cone and preferentially localized throughout the front meshwork of the cone where branched actin filaments were most concentrated. No myosin VI labeling was found adjacent to the membranes along the length of the cone or connecting neighboring cones. This method has potential to reveal important information about precise relationships between actin-binding proteins, membranes, and different types of actin structures.

As part of a study of cytoskeletal proteins involved in Drosophila embryonic development, we have undertaken the molecular analysis of a 140-kD ATP-sensitive actin-binding protein (Miller, K. G., C. M. Field, and B. M. Alberts. 1989. J. Cell Biol. 109:2963-2975). Analysis of cDNA clones encoding this protein revealed that it represents a new class of unconventional myosin heavy chains. The amino-terminal two thirds of the protein comprises a head domain that is 29-33% identical (60-65% similar) to other myosin heads, and contains ATP-binding, actin-binding and calmodulin/myosin light chain-binding motifs. The carboxy-terminal tail has no significant similarity to other known myosin tails, but does contain a approximately 100-amino acid region that is predicted to form an alpha-helical coiled-coil. Since the unique gene that encodes this protein maps to the polytene map position 95F, we have named the new gene Drosophila 95F myosin heavy chain (95F MHC). The expression profile of the 95F MHC gene is complex. Examination of multiple cDNAs reveals that transcripts are alternatively spliced and encode at least three protein isoforms; in addition, a fourth isoform is detected on Western blots. Developmental Northern and Western blots show that transcripts and protein are present throughout the life cycle, with peak expression occurring during mid-embryogenesis and adulthood. Immunolocalization in early embryos demonstrates that the protein is primarily located in a punctate pattern throughout the peripheral cytoplasm. Most cells maintain a low level of protein expression throughout embryogenesis, but specific tissues appear to contain more protein. We speculate that the 95F MHC protein isoforms are involved in multiple dynamic processes during Drosophila development.

Myosin VI is an actin-based motor that has been implicated in many cellular processes. Studies in vertebrates have demonstrated that animals lacking this ubiquitously expressed myosin are viable. However in Drosophila, myosin VI loss of function has been thought to be lethal. We show here that complete loss of myosin VI is not lethal in flies and that the previously reported lethality of the null mutation (jar322) is most likely due to deletion of a neighboring gene. Maternally provided myosin VI does not account for the survival of myosin VI null animals. Mutant animals are recovered at a lower than expected Mendelian frequency, suggesting that myosin VI participates in processes which contribute to normal development, but its participation is not essential.

Drosophila melanogaster bristle development is dependent on actin assembly, and prominent actin bundles form against the elongating cell membrane, giving the adult bristle its characteristic grooved pattern. Previous work has demonstrated that several actin-regulating proteins are required to generate normal actin bundles. Here we have addressed how two actin regulators, capping protein, a barbed end binding protein, and the Arp2/3 complex, a potent actin assembly nucleator, function to generate properly organized bundles. As predicted from studies in motile cells, we find that capping protein and the Arp2/3 complex act antagonistically to one another during bristle development. However, these proteins do not primarily act directly on bundles, but rather on a dynamic population of actin filaments that are not part of the bundles. These nonbundle filaments, termed snarls, play an important role in determining the number and spacing of the actin bundles. Reduction of capping protein leads to an increase in snarls, which prevents actin bundles from properly attaching to the membrane. Conversely, loss of an Arp2/3 complex component leads to a loss of snarls and accumulation of excess membrane-attached bundles. These results indicate that in nonmotile cells dynamic actin filaments can function to regulate the positioning of stable actin structures. In addition, our results suggest that the Arpc1 subunit may have an additional function, independent of the rest of the Arp2/3 complex.

Here, we demonstrate a new function of myosin VI using observations of Drosophila spermatid individualization in vivo. We find that myosin VI stabilizes a branched actin network in actin structures (cones) that mediate the separation of the syncytial spermatids. In a myosin VI mutant, the cones do not accumulate F-actin during cone movement, whereas overexpression of myosin VI leads to bigger cones with more F-actin. Myosin subfragment 1-fragment decoration demonstrated that the actin cone is made up of two regions: a dense meshwork at the front and parallel bundles at the rear. The majority of the actin filaments were oriented with their pointed ends facing in the direction of cone movement. Our data also demonstrate that myosin VI binds to the cone front using its motor domain. Fluorescence recovery after photobleach experiments using green fluorescent protein-myosin VI revealed that myosin VI remains bound to F-actin for minutes, suggesting its role is tethering, rather than transporting cargo. We hypothesize that myosin VI protects the actin cone structure either by cross-linking actin filaments or anchoring regulatory molecules at the cone front. These observations uncover a novel mechanism mediated by myosin VI for stabilizing long-lived actin structures in cells.

Myosin VI is a member of a superfamily of actin-based motors with at least 18 different sub-types or classes. Myosins are best known as proteins that use ATP-hydrolysis-mediated conformational changes to move along actin filaments. Because of this property, some myosins, including myosins I, V, and VI, are thought to be transporters of vesicle or protein cargoes. Myosin VI has been implicated in many seemingly different processes through functional studies in flies, worms and mammals. In several cases, its role is not easily explained by transport along actin. In addition, some of the biochemical and biophysical properties of myosin VI suggest other mechanisms of action. In this review, we summarize recent data that suggest diverse functions for myosin VI and offer an explanation for how myosin VI may function similarly in all of them. We hypothesize that the main function of myosin VI is to bind tightly to actin, stabilizing actin cytoskeletal structures and linking actin structures to membranes and protein complexes.

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.

In order to better understand the mechanism of sperm individualization during spermatogenesis in Drosophila melanogaster, we have developed an in vitro culture system in which we can perform live observation of individualization in isolated cysts. The whole process of individualization, during which a bundle of 64 syncytial spermatids is separated into individual sperm, takes place in these cultures. Individualization complexes, which consist of 64 cones of actin that assemble around the sperm nuclei, move to the basal end of the tails, forming a characteristic `cystic bulge9 that contains an accumulation of cytoplasm, syncytial membrane and vesicles. The cystic bulge is the site of membrane remodeling and its movement was used to follow the progress of individualization. The speed of cystic bulge movement is fairly constant along the length of the cyst. Actin drugs, but not microtubule drugs inhibit cystic bulge movement, suggesting that the movement requires proper actin dynamics but not microtubules. GFP-tagged actin was expressed in the cyst and fluorescence recovery after photobleaching was monitored using confocal microscopy to analyze actin dynamics in cones. Actin turns over throughout the cone, with that at the leading edge of the cones turning over with slightly faster kinetics. Actin does not treadmill from the front to the back of the cone. Actin in moving actin cones turns over in about 12 minutes, although prior to onset of movement, turnover is much slower. Visualization of membrane using FM1-43 reveals that the cystic bulge has an extremely complicated series of membrane invaginations and the transition from syncytial to individualized spermatids occurs at the front of the actin cones. We also suggest that endocytosis and exocytosis might not be important for membrane remodeling. This system should be suitable for analysis of defects in male sterile mutants and for investigating other steps of spermatogenesis.

Myosins are actin-activated ATPases that are able to translocate along actin filaments using energy derived from ATP hydrolysis. Non-muscle cells contain conventional myosins, which are similar in sequence and structure to muscle myosin, and a number of unconventional myosins whose head sequences are similar but tail sequences are unrelated to conventional myosins. The myosin superfamily currently consists of nine classes; Drosophila 95F is an unconventional myosin and the original member of class VI, which includes a homologue found in pig kidney. Some unconventional myosins have been suggested as mediators of some types of intracellular transport, but there is little direct evidence for this function (but see ref. 6). We have observed transport of cytoplasmic particles in live Drosophila embryos in three dimensions using computational optical sectioning microscopy. We present here evidence that this transport is actin-based, ATP-dependent and catalysed by one such unconventional myosin, the 95F myosin. This is, to our knowledge, the first direct observation of transport catalysed by an unconventional myosin in living cells.

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.

The American Society for Cell Biology (ASCB) recently published a short collection of essays entitled Career Advice for Life Scientists. This useful book is drawn from the Women in Cell Biology (WICB) column in the ASCB monthly newsletter. The WICB column has long been a highlight of the newsletter for many readers. It routinely discusses issues of interest to life scientists. Although a few essays both in the columns and in the compilation deal specifically with issues related to being a woman in science, most provide valuable advice and insight equally applicable to men and women. The editors of the book (Elizabeth Marincola, Executive Director of the Society, and the members of WICB) selected the columns that they judged to give the best advice in a number of areas important for success in science, including promotion strategies, lab management, and career–family balance. Each essay is only a few pages long, providing a succinct summary of relevant and useful information. The short format means that one or two essays can be read in 10–15 min, so they fit nicely as a break between other more lengthy tasks. Most essays are structured to give practical advice for everyday living as a life scientist—and most accomplish this goal remarkably well. This book should be required reading for lab heads and graduate students alike.

We have identified partial loss of function mutations in class VI unconventional myosin, 95F myosin, which results in male sterility. During spermatogenesis the germ line precursor cells undergo mitosis and meiosis to form a bundle of 64 spermatids. The spermatids remain interconnected by cytoplasmic bridges until individualization. The process of individualization involves the formation of a complex of cytoskeletal proteins and membrane, the individualization complex (IC), around the spermatid nuclei. This complex traverses the length of each spermatid resolving the shared membrane into a single membrane enclosing each spermatid. We have determined that 95F myosin is a component of the IC whose function is essential for individualization. In wild-type testes, 95F myosin localizes to the leading edge of the IC. Two independent mutations in 95F myosin reduce the amount of 95F myosin in only a subset of tissues, including the testes. This reduction of 95F myosin causes male sterility as a result of defects in spermatid individualization. Germ line transformation with the 95F myosin heavy chain cDNA rescues the male sterility phenotype. IC movement is aberrant in these 95F myosin mutants, indicating a critical role for 95F myosin in IC movement. This report is the first identification of a component of the IC other than actin. We propose that 95F myosin is a motor that participates in membrane reorganization during individualization.

Myosin VI can move along actin filaments to serve as a transport motor. It is also thought to anchor vesicles or proteins to actin. How these two diverse activities, which require very different modes of interaction with actin, are mediated is not understood. Using single molecule observations, Altman et al. (2004)([this issue of Cell]) demonstrate that load applied to myosin VI can convert this motor from a transporter to an anchor.

The 95F myosin, a class VI unconventional myosin, associates with particles in the cytoplasm of the Drosophila syncytial blastoderm and is required for the ATP- and F-actin-dependent translocation of these particles. The particles undergo a cell cycle-dependent redistribution from domains that surround each nucleus in interphase to transient membrane invaginations that provide a barrier between adjacent spindles during mitosis. When 95F myosin function is inhibited by antibody injection, profound defects in syncytial blastoderm organization occur. This disorganization is seen as aberrant nuclear morphology and position and is suggestive of failures in cytoskeletal function. Nuclear defects correlate with gross defects in the actin cytoskeleton, including indistinct actin caps and furrows, missing actin structures, abnormal spacing of caps, and abnormally spaced furrows. Three-dimensional examination of embryos injected with anti-95F myosin antibody reveals that actin furrows do not invaginate as deeply into the embryo as do normal furrows. These furrows do not separate adjacent mitoses, since microtubules cross over them. These inappropriate microtubule interactions lead to aberrant nuclear divisions and to the nuclear defects observed. We propose that 95F myosin function is required to generate normal actin-based transient membrane furrows. The motor activity of 95F myosin itself and/or components within the particles transported to the furrows by 95F myosin may be required for normal furrows to form.

In this summary of methods, we have attempted to present a series of protocols relevant to the study of proteins that associate with each other inside cells. These techniques have found their main application, in our hands, to cytoskeletal structures, but the utility of these techniques is limited to this application. As we learn more about the physiology and organization of eukaryotic cells, information from a variety of studies and systems indicates that many processes occur in a highly spatially organized manner in cells, using multiprotein complexes. The techniques described here will find application to the study of how organization and associations between proteins contribute to many of cellular processes.

In the syncytial blastoderm stage of Drosophila embryogenesis, dome-shaped actin "caps" are observed above the interphase nuclei. During mitosis, this actin rearranges to participate in the formation of pseudocleavage furrows, transient membranous invaginations between dividing nuclei. Embryos laid by homozygous sponge mothers lack these characteristic actin structures, but retain other actin associated structures and processes. Our results indicate that the sponge product is specifically required for the formation of actin caps and metaphase furrows. The specificity of the sponge phenotype permits dissection of both the process of actin cap formation and the functions of actin caps and metaphase furrows. Our data demonstrate that the distribution of actin binding protein 13D2 is unaffected in sponge embryos and suggest that 13D2 is upstream of actin in cortical cap assembly. Although actin caps and metaphase furrows have been implicated in maintaining the fidelity of nuclear division and the positions of nuclei within the cortex, our observations indicate that these structures are dispensible during the early syncytial blastoderm cell cycles. A later requirement for actin metaphase furrows in preventing the nucleation of mitotic spindles between inappropriate centrosomes is observed. Furthermore, the formation of actin caps and metaphase furrows is not a prerequisite for the formation of the hexagonal array of actin instrumental in the conversion of the syncytial embryo into a cellular blastoderm.

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.

The in vitro initiation site for RNA polymerase I on the mouse rRNA gene was identified using a new method that is generally applicable to the study of other eukaryotic transcripts. First, the 5' end of mouse rRNA was located to an ApC . . . by high resolution S1 nuclease mapping. Dinucleotide primers were then used in transcription reactions to demonstrate that this position is the actual de novo initiation site, and not a rapid RNA processing site. For this analysis, initiation was inhibited by reduced rXTP concentration, and, upon supplementation with various dinucleotides, only ApC stimulated correct synthesis. Independently confirming its role as the initiating nucleotide, ATP was shown to be required at a much higher concentration than the other rXTPs for RNA initiation, but not for elongation. These results also demonstrate a marked sequence conservation of rRNA initiation sites between the mouse and frog, two species that violate the general rule of species specificity in RNA polymerase I initiation. Extending these studies to RNA polymerase III, the initiation site for 5 S RNA can be similarly located by dinucleotide analysis and confirmed from the concentration requirements of each rXTP. In addition to allowing initiation at suboptimal rXTP concentration, dinucleotide primers can also circumvent the need for a factor normally required for initiation, suggesting their potential value in dissecting the mechanism of eukaryotic transcription.

Oligodendroglia isolated in bulk from dissected white matter of lamb or calf brain can now be maintained in culture for several days. The cells remain intact and viable during this time. The in vitro incorporation of various radiolabeled precursors into lipids or proteins was examined, and its was found that oligodendroglia synthesize the lipids which are predominant in brain, and especially in myelin. They actively synthesize cholesterol, cerebrosides, sulfatides, and all of the phospholipids, especially phosphatidylcholine. In addition, they produce a variety of radiolabeled proteins, with those of high molecular weight exhibiting the most radioactivity. Thus the cells are metabolically very active in vitro and can be used as a model system to study turnover of lipids in the cell, as well as the synthesis of myelin-specific components.

Using kinetoplast DNA networks as a substrate in a decatenation assay, we have purified to apparent homogeneity a type II DNA topoisomerase from HeLa cell nuclei. The most pure preparations contain a single polypeptide of 172,000 daltons as determined by sodium dodecyl sulfate-gel electrophoresis. The molecular weight of the native protein, based on sedimentation and gel filtration analyses, is estimated to be 309,000. These results suggest that the enzyme is a dimer of 172,0090-dalton subunits. The enzyme is a type II topoisomerase as demonstrated by its ability to change the linking number of DNA circles in steps of two and to decatenate or unknot covalently closed DNA circles. No gyrase activity is detectable. ATP is required for the relaxation, decatenation, and unknotting of DNA, and a DNA-dependent ATPase activity is present in the most pure fractions. ATP is hydrolyzed to ADP in this properties to T4 DNA topoisomerase (Liu, L. F., Liu, C. C., and Alberts, B. M. (1979) Nature 281, 456-461).

By using F-actin affinity chromatography columns to select proteins solely by their ability to bind to actin filaments, we have identified and partially purified greater than 40 proteins from early Drosophila embryos. These proteins represent approximately 0.5% of the total protein present in soluble cell extracts, and 2 mg are obtained by chromatography of an extract from 10 g of embryos. As judged by immunofluorescence of fixed embryos, 90% of the proteins that we have detected in F-actin column eluates are actin-associated in vivo (12 of 13 proteins tested). The distributions of antigens observed suggest that groups of these proteins cooperate in generating unique actin structures at different places in the cell. These structures change as cells progress through the cell cycle and as they undergo the specializations that accompany development. The variety of different spatial localizations that we have observed in a small subset of the total actin-binding proteins suggests that the actin cytoskeleton is a very complex network of interacting proteins.