A Unique and Scalable Model for Increasing Research Engagement, STEM Persistence, and Entry into Doctoral Programs.

Low persistence in science majors and limited participation in high-impact research experiences contribute to the nationwide underrepresentation of minorities in the science workforce, particularly jobs requiring a graduate degree. The Program for Excellence in Education and Research in the Sciences (PEERS) is an academic support program at the University of California, Los Angeles (UCLA) that supports first- and second-year science majors from underrepresented and underserved backgrounds to maximize student success and science, technology, engineering, and mathematics (STEM) persistence. Here, we evaluate the success of PEERS through data from the UCLA registrar, student surveys, and longitudinal tracking of student outcomes. Results show that PEERS students have significantly higher participation rates in undergraduate research, despite PEERS having no formal research component. Importantly, PEERS students were seven times as likely to enroll in PhD programs, and twice as likely to enroll in MD programs compared with propensity-matched controls. Combined results show that increased success of PEERS students in their first 2 years as science majors resulted in improved outcomes later in their undergraduate studies and had tangible impacts on subsequent educational trajectories that will increase participation of underrepresented groups in high-skill STEM careers.

Increasing persistence in undergraduate science majors: a model for institutional support of underrepresented students.

The 6-yr degree-completion rate of undergraduate science, technology, engineering, and mathematics (STEM) majors at U.S. colleges and universities is less than 40%. Persistence among women and underrepresented minorities (URMs), including African-American, Latino/a, Native American, and Pacific Islander students, is even more troubling, as these students leave STEM majors at significantly higher rates than their non-URM peers. This study utilizes a matched comparison group design to examine the academic achievement and persistence of students enrolled in the Program for Excellence in Education and Research in the Sciences (PEERS), an academic support program at the University of California, Los Angeles, for first- and second-year science majors from underrepresented backgrounds. Results indicate that PEERS students, on average, earned higher grades in most "gatekeeper" chemistry and math courses, had a higher cumulative grade point average, completed more science courses, and persisted in a science major at significantly higher rates than the comparison group. With its holistic approach focused on academics, counseling, creating a supportive community, and exposure to research, the PEERS program serves as an excellent model for universities interested in and committed to improving persistence of underrepresented science majors and closing the achievement gap.

A Myo6 mutation destroys coordination between the myosin heads, revealing new functions of myosin VI in the stereocilia of mammalian inner ear hair cells.

Myosin VI, found in organisms from Caenorhabditis elegans to humans, is essential for auditory and vestibular function in mammals, since genetic mutations lead to hearing impairment and vestibular dysfunction in both humans and mice. Here, we show that a missense mutation in this molecular motor in an ENU-generated mouse model, Tailchaser, disrupts myosin VI function. Structural changes in the Tailchaser hair bundles include mislocalization of the kinocilia and branching of stereocilia. Transfection of GFP-labeled myosin VI into epithelial cells and delivery of endocytic vesicles to the early endosome revealed that the mutant phenotype displays disrupted motor function. The actin-activated ATPase rates measured for the D179Y mutation are decreased, and indicate loss of coordination of the myosin VI heads or 'gating' in the dimer form. Proper coordination is required for walking processively along, or anchoring to, actin filaments, and is apparently destroyed by the proximity of the mutation to the nucleotide-binding pocket. This loss of myosin VI function may not allow myosin VI to transport its cargoes appropriately at the base and within the stereocilia, or to anchor the membrane of stereocilia to actin filaments via its cargos, both of which lead to structural changes in the stereocilia of myosin VI-impaired hair cells, and ultimately leading to deafness.

Precise positioning of myosin VI on endocytic vesicles in vivo.

Myosin VI has been studied in both a monomeric and a dimeric form in vitro. Because the functional characteristics of the motor are dramatically different for these two forms, it is important to understand whether myosin VI heavy chains are brought together on endocytic vesicles. We have used fluorescence anisotropy measurements to detect fluorescence resonance energy transfer between identical fluorophores (homoFRET) resulting from myosin VI heavy chains being brought into close proximity. We observed that, when associated with clathrin-mediated endocytic vesicles, myosin VI heavy chains are precisely positioned to bring their tail domains in close proximity. Our data show that on endocytic vesicles, myosin VI heavy chains are brought together in an orientation that previous in vitro studies have shown causes dimerization of the motor. Our results are therefore consistent with vesicle-associated myosin VI existing as a processive dimer, capable of its known trafficking function.

Binding of internalized receptors to the PDZ domain of GIPC/synectin recruits myosin VI to endocytic vesicles.

Myosin VI (myo6) is the only actin-based molecular motor that translocates along actin filaments toward the minus end. Myo6 participates in two steps of endocytic trafficking; it is recruited to both clathrin-coated pits and to ensuing uncoated endocytic vesicles (UCV). Although there is evidence suggesting that the PDZ adaptor protein GIPC/synectin is involved in the association of myo6 with UCV, the recruitment mechanism is unknown. We show that GIPC/synectin is required for both internalization of cell surface receptors and for coupling of myo6 to UCV. This coupling occurs via a mechanism wherein engagement of the GIPC/synectin PDZ domain by C termini of internalized receptors facilitates in trans myo6 binding to the GIPC/synectin C terminus located outside of the PDZ domain. Analysis of megalin, a prototypical GIPC/synectin-binding receptor, revealed that deletion of its PDZ-binding motif drastically reduced GIPC/synectin and myo6 recruitment to UCV. Furthermore, interaction with GIPC/synectin was required for megalin's function, as megalin was mistargeted in the renal proximal tubules of GIPC/synectin-null mice and these mice exhibited proteinuria, a condition consistent with defective megalin trafficking.

Myosin VI altered at threonine 406 stabilizes actin filaments in vivo.

Myosin VI is a minus-end directed actin-based molecular motor implicated in uncoated endocytic vesicle transport. Recent kinetic studies have shown that myosin VI displays altered ADP release kinetics under different load conditions allowing myosin VI to serve alternately as a transporter or as an actin tether. We theorized that one potential regulatory event to modulate between these kinetic choices is phosphorylation at a conserved site, threonine 406 (T406) in the myosin VI motor domain. Alterations mimicking the phosphorylated (T406E) and dephosphorylated state (T406A) were introduced into a GFP-myosin VI fusion (GFP-M6). Live cell imaging revealed that GFP-M6(T406E) expression changed the path myosin VI took in its transport of uncoated endocytic vesicles. Rather than routing vesicles inwards as seen in GFP-M6 and GFP-M6(T406A) expressing cells, GFP-M6(T406E) moved vesicles into clusters at distinct peripheral sites. GFP-M6(T406E) expression also increased the density of the actin cytoskeleton. Filaments were enriched at the vesicle cluster sites. This was not due to a gross redistribution of the actin polymerization machinery. Instead the filament density correlated to the fixed positioning of GFP-M6(T406E)-associated vesicles on F-actin, leading to inhibition of actin depolymerization. Our study suggests that phosphorylation at T406 changes the nature of myosin VI's interaction with actin in vivo.

Physical and functional interaction between protocadherin 15 and myosin VIIa in mechanosensory hair cells.

Hair cells of the mammalian inner ear are the mechanoreceptors that convert sound-induced vibrations into electrical signals. The molecular mechanisms that regulate the development and function of the mechanically sensitive organelle of hair cells, the hair bundle, are poorly defined. We link here two gene products that have been associated with deafness and hair bundle defects, protocadherin 15 (PCDH15) and myosin VIIa (MYO7A), into a common pathway. We show that PCDH15 binds to MYO7A and that both proteins are expressed in an overlapping pattern in hair bundles. PCDH15 localization is perturbed in MYO7A-deficient mice, whereas MYO7A localization is perturbed in PCDH15-deficient mice. Like MYO7A, PCDH15 is critical for the development of hair bundles in cochlear and vestibular hair cells, controlling hair bundle morphogenesis and polarity. Cochlear and vestibular hair cells from PCDH15-deficient mice also show defects in mechanotransduction. Together, our findings suggest that PCDH15 and MYO7A cooperate to regulate the development and function of the mechanically sensitive hair bundle.

The unconventional myosin-VIIa associates with lysosomes.

Mutations in the myosin-VIIa (MYO7a) gene cause human Usher disease, characterized by hearing impairment and progressive retinal degeneration. In the retina, myosin-VIIa is highly expressed in the retinal pigment epithelium, where it plays a role in the positioning of melanosomes and other digestion organelles. Using a human cultured retinal pigmented epithelia cell line, ARPE-19, as a model system, we have found that a population of myosin-VIIa is associated with cathepsin D- and Rab7-positive lysosomes. Association of myosin-VIIa with lysosomes was Rab7 independent, as dominant negative and dominant active versions of Rab7 did not disrupt myosin-VIIa recruitment to lysosomes. Association of myosin-VIIa with lysosomes was also independent of the actin and microtubule cytoskeleton. Myosin-VIIa copurified with lysosomes on density gradients, and fractionation and extraction experiments suggested that it was tightly associated with the lysosome surface. These studies suggest that myosin-VIIa is a lysosome motor.

Keap1 regulates the oxidation-sensitive shuttling of Nrf2 into and out of the nucleus via a Crm1-dependent nuclear export mechanism.

Keap1 is a negative regulator of Nrf2, a transcription factor essential for antioxidant response element (ARE)-mediated gene expression. We find that Keap1 sequesters Nrf2 in the cytoplasm, not by docking it to the actin cytoskeleton but instead through an active Crm1/exportin-dependent nuclear export mechanism. Deletion and mutagenesis studies identified a nuclear export signal (NES) in the intervening region of Keap1 comprised of hydrophobic leucine and isoleucine residues in agreement with a traditional NES consensus sequence. Mutation of the hydrophobic amino acids resulted in nuclear accumulation of both Keap1 and Nrf2, as did treatment with the drug leptomycin B, which inactivates Crm1/exportin. ARE genes were partially activated under these conditions, suggesting that additional oxidation-sensitive elements are required for full activation of the antioxidant response. Based on these data, we propose a new model for regulation of Nrf2 by Keap1. Under normal conditions, Keap1 and Nrf2 are complexed in the cytoplasm where they are targeted for degradation. Oxidative stress inactivates Keap1's NES, allowing entry of both Keap1 and Nrf2 into the nucleus and transcriptional transactivation of ARE genes.

Regulation of myosin-VI targeting to endocytic compartments.

Myosin-VI has been implicated in endocytic trafficking at both the clathrin-coated and uncoated vesicle stages. The identification of alternative splice forms led to the suggestion that splicing defines the vesicle type to which myosin-VI is recruited. In contrast to this hypothesis, we find that in all cell types examined, myosin-VI is associated with uncoated endocytic vesicles, regardless of splice form. GIPC, a PDZ-domain containing adapter protein, co-assembles with myosin-VI on these vesicles. Myosin-VI is only recruited to clathrin-coated vesicles in cells that express high levels of Dab2, a clathrin-binding adapter protein. Overexpression of Dab2 is sufficient to reroute myosin-VI to clathrin-coated pits in cells where myosin-VI is normally associated with uncoated vesicles. In normal rat kidney (NRK) cells, which express high endogenous levels of Dab2, splicing of the globular tail domain further modulates targeting of ectopically expressed myosin-VI. Although myosin-VI can be recruited to clathrin-coated pits, we find no requirement for myosin-VI motor activity in endocytosis in NRK cells. Instead, our data suggest that myosin-VI recruitment to clathrin-coated pits may be an early step in the recruitment of GIPC to the vesicle surface.

Myosin VI regulates endocytosis of the cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-regulated Cl(-) channel expressed in the apical plasma membrane in fluid-transporting epithelia. Although CFTR is rapidly endocytosed from the apical membrane of polarized epithelial cells and efficiently recycled back to the plasma membrane, little is known about the molecular mechanisms regulating CFTR endocytosis and endocytic recycling. Myosin VI, an actin-dependent, minus-end directed mechanoenzyme, has been implicated in clathrin-mediated endocytosis in epithelial cells. The goal of this study was to determine whether myosin VI regulates CFTR endocytosis. Endogenous, apical membrane CFTR in polarized human airway epithelial cells (Calu-3) formed a complex with myosin VI, the myosin VI adaptor protein Disabled 2 (Dab2), and clathrin. The tail domain of myosin VI, a dominant-negative recombinant fragment, displaced endogenous myosin VI from interacting with Dab2 and CFTR and increased the expression of CFTR in the plasma membrane by reducing CFTR endocytosis. However, the myosin VI tail fragment had no effect on the recycling of endocytosed CFTR or on fluid-phase endocytosis. CFTR endocytosis was decreased by cytochalasin D, an actin-filament depolymerizing agent. Taken together, these data indicate that myosin VI and Dab2 facilitate CFTR endocytosis by a mechanism that requires actin filaments.

Uncoated endocytic vesicles require the unconventional myosin, Myo6, for rapid transport through actin barriers.

After clathrin-mediated endocytosis, clathrin removal yields an uncoated vesicle population primed for fusion with the early endosome. Here we present the first characterization of uncoated vesicles and show that myo6, an unconventional myosin, functions to move these vesicles out of actin-rich regions found in epithelial cells. Time-lapse microscopy revealed that myo6-associated uncoated vesicles were motile and exhibited fusion and stretching events before endosome delivery, processes that were dependent on myo6 motor activity. In the absence of myo6 motor activity, uncoated vesicles remained trapped in the actin mesh, where they exhibited Brownian-like motion. Exit from the actin mesh occurred by a slow diffusion-based mechanism, delaying transferrin trafficking to the early endosome. Expression of a myo6 mutant that bound tightly to F-actin produced immobilized vesicles and blocked trafficking. Depolymerization of the actin cytoskeleton rescued this block and specifically accelerated transferrin delivery to the early endosome without affecting earlier steps in endocytosis. Therefore actin is a physical barrier impeding uncoated vesicle trafficking, and myo6 is recruited to move the vesicles through this barrier for fusion with the early endosome.

Keap1 in adhesion complexes.

Cell adhesion complexes are sensors that interact with the extracellular environment and allow for the transmission of signals found outside the cell across the plasma membrane to the cell interior. Keap1 is a newly identified component of cell adhesion complexes. We investigated Keap1's association with these complexes in diverse tissues and cell types. Keap1 is present in focal adhesion (FA)-like assemblies in kidney proximal tubule cells where it colocates with actin. In liver, Keap1 is found in the adherens junctions (AJ) and at the base of the bile canaliculi. To study Keap1's involvement in both the integrin-based FA and the cadherin-based AJ, we induced formation of these complexes in fibroblasts, using a serum starvation followed by a serum supplementation method. When compared with vinculin, a component of all FA, we found that Keap1 assembles only in the peripheral FA. Within the peripheral FA, Keap1 was present in distinct foci along the length of the FA and these foci were different from vinculin, talin, paxillin, and phospho-tyrosine rich regions of the FA. Unlike most FA components, Keap1 was also recruited to the newly formed AJ. As Keap1 homologues are actin-bundling proteins, we hypothesize that Keap1's function is to bundle F-actin within these diverse types of cell adhesion components.

Myosin VI: two distinct roles in endocytosis.

Actin is found at the cortex of the cell where endocytosis occurs, but does it play a role in this essential process? Recent studies on the unconventional myosin, myosin VI, an actin-based molecular motor, provide compelling evidence that this myosin and therefore actin is involved in two distinct steps of endocytosis in higher eukaryotes: the formation of clathrin-coated vesicles and the movement of nascent uncoated vesicles from the actin-rich cell periphery to the early endosome. Three distinct adapter proteins--GIPC, Dab2 and SAP97--that associate with the cargo-binding tail domain of myosin VI have been identified. These proteins may recruit myosin VI to its sites of action.

Myo6 facilitates the translocation of endocytic vesicles from cell peripheries.

Immunolocalization studies in epithelial cells revealed myo6 was associated with peripherally located vesicles that contained the transferrin receptor. Pulse-chase experiments after transferrin uptake showed that these vesicles were newly uncoated endocytic vesicles and that myo6 was recruited to these vesicles immediately after uncoating. GIPC, a putative myo6 tail binding protein, was also present. Myo6 was not present on early endosomes, suggesting that myo6 has a transient association with endocytic vesicles and is released upon early endosome fusion. Green fluorescent protein (GFP) fused to myo6 as well as the cargo-binding tail (M6tail) alone targeted to the nascent endocytic vesicles. Overexpression of GFP-M6tail had no effect on a variety of organelle markers; however, GFP-M6tail displaced the endogenous myo6 from nascent vesicles and resulted in a significant delay in transferrin uptake. Pulse-chase experiments revealed that transferrin accumulated in uncoated vesicles within the peripheries of transfected cells and that Rab5 was recruited to the surface of these vesicles. Given sufficient time, the transferrin did traffic to the perinuclear sorting endosome. These data suggest that myo6 is an accessory protein required for the efficient transportation of nascent endocytic vesicles from the actin-rich peripheries of epithelial cells, allowing for timely fusion of endocytic vesicles with the early endosome.

Expression of myosin VI within the early endocytic pathway in adult and developing proximal tubules.

Myosin VI is a reverse-direction molecular motor implicated in membrane transport events. Because myosin VI is most highly expressed in the kidney, we investigated its renal localization by using high-resolution immunocytochemical and biochemical methods. Indirect immunofluorescence microscopy revealed myosin VI at the base of the brush border in proximal tubule cells. Horseradish peroxidase uptake studies, which labeled endosomes, and double staining for clathrin adapter protein-2 showed that myosin VI was closely associated with the intermicrovillar (IMV) coated-pit region of the brush border. Localization of myosin VI to the IMV region was confirmed at the electron microscopic level by colloidal gold labeling of ultrathin cryosections. In addition, antigen retrieval demonstrated a small but significant pool of myosin VI on the microvilli. To confirm the association of myosin VI with the IMV compartment, these membranes were separated from other membrane compartments by using 15-25% OptiPrep density gradients. Immunoblotting of the gradient fractions confirmed that myosin VI was enriched with markers for the IMV microdomain of the brush border, suggesting that myosin VI associates with proteins in this compartment. Finally, we examined the expression of myosin VI during nephron development. We found myosin VI present in a diffuse cytoplasmic pattern at stage II (S-shaped body phase) and that it was only redistributed fully to the brush border in the stage IV nephron. These studies support a model for myosin VI function in the endocytic process of the proximal tubule.

A human homologue of Drosophila kelch associates with myosin-VIIa in specialized adhesion junctions.

Mutations in myosin-VIIa are responsible for the deaf-blindness, Usher disease. Myosin-VIIa is also highly expressed in testis, where it is associated with specialized adhesion plaques termed ectoplasmic specializations (ES) that form between Sertoli cells and germ cells. To identify new roles for myosin-VIIa, we undertook a yeast two-hybrid screen to identify proteins associated with myosin-VIIa in the ES. We identified Keap1, a human homologue of the Drosophila ring canal protein, kelch. The kelch-repeats in the C-terminus of human Keap1 associate with the SH3 domain of myosin-VIIa. Immunolocalization studies revealed that Keap1 is present with myosin-VIIa in the actin bundles of the ES. Myosin-VIIa and Keap1 copurify with ES and colocate with each other and with F-actin at the electron microscopy level. Interestingly, in many epithelial cell types including cells derived from retina and inner ear, Keap1 is a component of focal adhesions and zipper junctions. Keap1 can target to the ES in the absence of myosin-VIIa, suggesting that Keap1 associates with other molecules in the adhesion plaque. Keap1 and myosin-VIIa overlapped in expression in the inner hair cells of the cochlea, suggesting that Keap1 may be a part of a family of actin-binding proteins that could be important for myosin-VIIa function in testis and inner ear.