Daniel A. Starr - US grants

Cell migration events occur throughout development as a part of a differentiation program to allow cells to form structures and perform specialized functions. It is important to determine the molecular mechanisms of cell migration to better understand both development and how a cancer cell migrates. Nuclear migration has been shown to be an important aspect of many cellular migration events during C elegans development. Because of its optical clarity and powerful genetics, C. elegans is an excellent model system in which to study cellular and nuclear migrations. In C. elegans, the P cells normally migrate to the dorsal cord where they eventually give rise to neurons and the vulva. Mutations in two genes, unc-83 or unc-84, block this migration by interfering with nuclear migration, leading to an adult worm that is uncoordinated and egg-laying defective. In addition, mutations in unc-84 and a third gene, anc-1, disrupt mechanisms controlling nuclear anchorage. In order to more fully understand the control and mechanisms of nuclear migration, and more generally, cellular migrations, experiments are proposed to characterize the roles these three genes play in nuclear migration. unc-83 and anc-1 (unc-84 is already cloned) will be cloned, and the lesions in our large collection of mutant alleles will be molecularly characterized. Then antibodies will be raised against these proteins to determine their sub-cellular localization. Additionally, genetic and molecular screens are proposed to identify other components involved in nuclear migration.

The position of the nucleus is carefully controlled in a wide variety of cell types. Nuclear migration plays a role in normal cell migration events and metastasis;defects in nuclear migration lead to the neurological disease Lissencephaly. Nuclear anchorage functions in the development of the neuro-muscular junction and may contribute to muscular dystrophy. A group of three conserved nuclear envelope proteins are required for proper nuclear positioning in C. elegans. Our objective is to characterize how these proteins function to control nuclear positioning and to identify other proteins that function with or in parallel to them. Our central hypothesis is that UNC-84 functions at the inner nuclear membrane to recruit UNC-83 and ANC-1 to the outer nuclear membrane. Together, they bridge the nuclear envelope to connect the nuclear matrix to the cytoskeleton. Our first aim will determine the topology of these three proteins using an in vivo protease protection assay and immuno-EM. Aim 2 will use molecular genetic techniques to test the central link of our model, the interaction between the SUN domain of UNC-84 and the KASH domains of UNC-83 and ANC-1. In aim 3 we expect to link UNC-83 to the cytoskeleton by identifying interacting partners through biochemical and molecular screens using essential portions of the novel domain of UNC-83. In aim 4 we take a genetic approach to identify additional proteins involved in nuclear positioning by cloning existing enhancer of unc-83 or unc-84 alleles. We will use genome-wide RNAi to screen for more enhancers. The ability to combine genetic, biochemical, and molecular approaches in a developmental system makes C, elegans a powerful system for these studies. Together, these studies will provide mechanistic insight into the fundamental problem of how the nucleus positions itself in the cytoplasm.

DESCRIPTION (provided by applicant: A wide variety of cellular processes, including fertilization, cell division, cell migration, and cell polarity, depend on nuclear migration events. Inner nuclear membrane SUN proteins and outer nuclear membrane KASH proteins couple nuclei to the cytoskeleton. Gaps remain in understanding how KASH-SUN bridges are formed and function. Specifically, the molecular mechanisms of how proteins are trafficked to the inner nuclear mem- brane, how microtubules and motors are coordinated to move nuclei, and how KASH and SUN proteins interact to connect cytoplasmic forces to nuclei remain unknown. Our hypothesis is that forces generated by microtubule motors in the cytoplasm are connected to the nucleus by a bridge of conserved KASH and SUN proteins. Understanding how forces are transferred across the nuclear envelope will allow us to elucidate mechanisms of how nuclei are positioned in a cell, how chromosomes are moved inside the nucleus, and how perturbations of these processes disrupt cell and developmental processes. Our model will be tested by three specific aims: (Aim 1) Elucidate mechanisms of inner nuclear membrane biogenesis. The current paradigm is that membrane proteins diffuse within the ER membrane to the nuclear envelope. Our preliminary data support an alternative active transport model for inner nuclear membrane trafficking, using a combination of the soluble nuclear import machinery, membrane-bound importins, and a Golgi trafficking intermediate. We hypothesize that multiple inner-nuclear-membrane-localization signals function to first actively transport UNC-84 from the peripheral ER to the nuclear envelope and to then mediate movement across the nuclear pore. (Aim 2) Deter- mine how kinesin, dynein, and microtubules function to move nuclei. Tug-of-war, interdependent regulation, and bi-directional movement are proposed models to explain how motors of opposite polarity function together to move a cargo. Our hypothesis is that kinesin-1 provides the force to move nuclei and that dynein mediates backwards movements and rolling to bypass roadblocks. We will distinguish between two models for how NOCA-1 regulates polarized microtubule arrays-by regulating either plus-end tip dynamics or nucleation of microtubules. (Aim 3) Determine how forces generated in the cytoplasm are coupled to the nucleus. Two models could explain the role of the KASH-SUN bridge in nuclear migration; they could serve simply as outer nuclear docking sites or, also as transducers of force across the nuclear envelope. We hypothesize that forces generated in the cytoplasm are directly linked to the nuclear lamina by KASH-SUN bridges. Our approach is innovative because it takes advantage of a C. elegans model with unique genetic and molecular strengths with the ability to film and quantify nuclear migration. The proposed research is significant because it is expected to (A) elucidate mechanisms of protein transport to the inner nuclear membrane, (B) elucidate mechanisms of bi- directional nuclear migration along polarized microtubules that will be applicable to other large cargos, and (C) determine how the forces that move nuclei are transferred across the nuclear envelope.

? DESCRIPTION (provided by applicant): Despite the importance of nuclear positioning for a wide variety of cell and developmental events, including cell migration, muscle development, CNS development, and cell polarity, the mechanisms of how nuclei move are poorly understood. It is unknown how nuclei switch between migration and anchorage or how some nuclei in normal development or metastasizing tumor cells squeeze through constricted spaces. The best-described mechanism to move nuclei involves a bridge across the nuclear envelope consisting of SUN and KASH proteins (the LINC complex) that couples nuclei to the cytoskeleton. However, SUN/KASH bridges are not required for many nuclear migration events, suggesting other, mostly unknown, mechanisms move nuclei. The goals here are to advance our mechanistic understanding of nuclear positioning by determining how nuclei switch between nuclear migration and anchorage, and to elucidate new mechanisms for nuclear migration through narrow openings. We hypothesize that differences in KASH partners contribute to SUN/KASH regulation and switching between migration and anchorage. Furthermore, a second, actin-mediated pathway functions in addition to SUN/KASH to move nuclei through constrained spaces. Our model will be tested by three specific aims: (1) Determine how SUN/KASH bridges are built and regulated to mediate switches between nuclear migration and nuclear anchorage. The in vivo phenotypic consequences and in vitro binding affinities of point mutations engineered into SUN/KASH interfaces will be examined. We will also test the hypothesis that cysteine-cysteine bonds regulate the switch between nuclear migration and anchorage using in vivo molecular engineering and mass spectrometry approaches. (2) Molecularly characterize how nuclei traverse spatially constricted cellular spaces. C. elegans P-cell nuclei flatten to migrate through a 150 nm space. We hypothesize three mechanisms function together to move nuclei: a KASH protein, UNC-83, recruits dynein to the surface of the nucleus to move nuclei toward the minus ends of microtubules, lamins regulate nuclear flattening, and actin-based pathways move nuclei thorough constricted spaces. (3) Elucidate mechanisms for how TOCA-1 and FLN-2 organize actin networks to move P-cell nuclei through narrow spaces. We identified actin-regulators toca-1 and fln-2 as functioning in a new pathway for nuclear migration. We will determine the intracellular localization and identify functional domains of TOCA-1 and FLN-2. We will also characterize how they mechanistically regulate actin organization. The proposed approach is innovative because of the combination of live imaging of nuclear movements in a developmental context and the powerful genetic approaches available in C. elegans. The proposed research is significant because it will lead to mechanistic insights for nuclear positioning. In summary, the research is expected to identify and characterize new and conserved mechanisms of nuclear migration, which will advance our understanding of basic cell and developmental processes related to human diseases, including cancer.

Many students from minority populations or disadvantaged backgrounds are underrepresented in PhD-level biomedical research, perhaps having decided not to enter PhD programs because they either lack academic preparation or fail to self-identify as research scientists. The goal of PREP at UC Davis (PREP@UCD) is to provide such students with the research, communication, analytical, and life skills required to excel in doctoral programs in the biomedical sciences. The University of California, Davis, has a broad base of biomedical research funded by the NIH, is committed to diversity through a host of campus-wide academic and cultural initiatives, and is dedicated to enriching undergraduate and graduate student education in the biomedical sciences, making it an ideal institution to host a PREP program. Moreover, UC Davis is actively pursuing status as a Hispanic-Serving Institution (HSI). Up to eight PREP scholars annually will participate in research (75%) and career development activities (25%) under the close guidance of a mentorship team consisting of research supervisors, a faculty advisor, the PI, and the program coordinator. Three specific aims are proposed to prepare PREP@UCD scholars for biomedical graduate programs. First, PREP will provide a mentored research environment where the scholars can develop their self-image as scientists. A recent study of PREP scholars found that success in graduate school requires that the student self-identifies as a scientist; spending a year performing individual, hypothesis-driven research will provide PREP@UCD scholars with the opportunity to do so. Second, PREP@UCD scholars will be trained in experimental skills and mentored in an active research laboratory. An individualized research prospectus will capture a hypothesis and line of research to expose the scholar to a variety of scientific approaches. Laboratory activities, academic coursework, training workshops and symposia will develop skills for gathering scientific information, working in a team, solving problems critically, and assessing research ethics and values. Third, PREP@UCD scholars will develop strong academic written and oral communication skills. Each scholar will spend 25% of his or her effort in preparing documents for graduate applications in biomedical sciences; enrolling in one senior-level course per quarter for enrichment; presenting research findings in poster and PowerPoint formats; participating in journal clubs to gain exposure to scientific literature and discourse; attending regional and national scientific conferences to network with other scholars; and participating in professional development workshops to prepare for the rigors of graduate school and career options beyond the PhD. It is expected that at least 75% of scholars will enter prestigious graduate programs upon completion of the PREP@UCD program. Moreover, the skills and scientific self-identity fostered during PREP@UCD will provide the scholars with a better chance of excelling in biomedical PhD or MD/PhD programs.

Project Summary Nuclear migration and anchorage are central to many cellular events. We uncovered a conserved network of nuclear envelope proteins and force generators that mediate nuclear positioning. LINC (linker of nucleoskele- ton and cytoskeleton) complexes, which we discovered, maintain nuclear envelope architecture, mark the surface of nuclei distinctly from the contiguous ER, and were instrumental in the early evolution of eukaryotes. We address four gaps in our knowledge of the mechanisms regulating nuclear positioning. (1) How is the developmental switch between nuclear migration and anchorage mediated? We hypothesize that different LINC complexes are required for a nucleus to switch from migrating to being anchored. We propose that an intermolecular disulfide bond, which could be regulated by protein disulfide isomerases and/or the AAA+ ATPase torsin, is central to the switch. We further hypothesize that LINC directly interacts with the outer nuclear membrane to optimize the transfer of forces across the nuclear envelope. (2) How are nuclei anchored in large syncytial cells? It is important for nuclei to be evenly spaced so that multi-nucleated syncytia are able to act as a single unit. We recently found that ANC-1 anchors syncytial nuclei and mitochondria through unknown, LINC-independent mechanisms, and hypothesize that ANC-1 organizes the cytoplasm through microtubules. (3) How do nuclei favor one microtubule motor over another at different stages of development? The KASH protein UNC-83 mediates nuclear movements toward plus or minus ends of microtubules at differ- ent stages of development. We hypothesize that the choice is regulated by alternative isoforms of UNC-83 that differentially activate kinesin-1 motor activity. (4) How do nuclei deform to migrate through narrow spaces? Our data support a model where LINC complexes function parallel to branched actin networks to deform nuclei as they squeeze through narrow constrictions. Our experimental system is innovative because we can view live nuclei throughout development, including a tissue where 139 nuclei are in a single hypodermal syncytium and a second tissue where nuclei migrate through narrow constrictions as a normal part of development. Further- more, we have developed reagents essential to our future plans, including an array of point mutants in LINC complexes that separate function, cell-specific markers, a tissue-specific auxin-induced degron system, and over ten mutant lines from a forward genetic screen for defects in nuclear migration through constrictions. To complement our C. elegans genetic approaches, we also collaborate to confirm our findings in mammalian tissue culture cells and an in vitro microtubule motor assay with TIRF microscopy. Our studies are expected to determine how LINC complexes are regulated at molecular and biophysical levels, how the outer nuclear membrane is involved in force transmission, how giant KASH proteins organize the global cytoskeleton and position organelles, how UNC-83 mediates the choice between dynein and kinesin-directed nuclear move- ments throughout development, and how actin helps nuclei squeeze through constricted spaces.