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.
The proposed research is relevant to public health because mutations in KASH, SUN, and other proteins that function to move and anchor nuclei have been shown to cause or to be linked to muscular dystrophies, ataxias, progeria, autism, and multiple cancers. Moreover, defects in nuclear migration contribute too many of the neuromuscular defects of these diseases. Thus, the proposed research is relevant to the part of the NIH's mission that fosters fundamental discoveries in basic cell and developmental biology with great potential for a positive impact on human health.
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