Title: Distinct Nuclear-Cytoskeletal LINCages Position the Nucleus for Homeostasis, Polarization and Migration
Description: Nuclear positioning occurs in different cellular contexts: from dividing yeast to more specialized cells like neuronal glial progenitor and skeletal muscle cells. Interestingly, abnormal nuclear positioning is associated with diseases such as muscular dystrophy where nuclei occupy a central rather than peripheral location. Moreover, rearward nuclear positioning is typical of migratory cells. Active nuclear movement in most cases involves coupling of cytoskeletal components with the nucleus by a group of transmembrane proteins in the nuclear envelope called the LINC (linker of nucleoskeleton and cytoskeleton) complex. It is composed of the inner nuclear membrane SUN (Sad1p, UNC-84) proteins associated with nuclear lamins and the outer nuclear membrane KASH (Klarsicht, ANC-1, Syne Homology) proteins, which interact with the cytoskeleton. In my thesis, the murine fibroblast cell line NIH3T3 was used as a model system to study nuclear positioning in states of active movement and static homeostatic positioning.

Nuclear positioning and centrosome reorientation are hallmarks of cell polarity in migrating fibroblasts. The Gundersen lab has established that the nucleus moves rearward to orient the centrosome in serum starved fibroblast monolayers stimulated by the serum-derived factor lysophosphatidic acid (LPA). LPA stimulates the GTPase Cdc42, which in turn activates the Cdc42 effector MRCK to phosphorylate myosin II and activate actin retrograde flow to move the nucleus to the rear. A second Cdc42 effector, Par6 functions with Par3 and dynein to maintain the centrosome in the cell centroid. The nucleus is moved rearward by the attachment of retrograde dorsal actin cables to the nucleus through transmembrane actin-associated nuclear (TAN) lines. TAN lines are composed linear arrays of the LINC complex proteins nesprin-2G (N2G) and SUN2 and dorsal actin cables. Disrupting TAN lines components blocks nuclear movement and efficient cell migration. Interestingly, TAN lines are analogous to other membrane adhesions, such as focal adhesions, in that they are transmembrane structures linked to the actin cytoskeleton and transmit force.

Given the large number of proteins composing structures such as focal adhesions, we predicted there would be additional components in TAN lines necessary for their formation and function. Thus, I set out to identify and study cytoplasmic factors required for TAN line formation and/or function during active nuclear positioning in fibroblast. A collaborator detected N2G as a hit in a yeast two-hybrid screen for FHOD1 interactors. FHOD1 is an actin regulator and belongs to the formin family. Like other formin family members, it has an FH2 actin binding domain, an FH1 domain and DID and DAD domains that interact to autoinhibit FHOD1. Unlike other formins, FHOD1 is not activated by GTPase binding and contains a second actin binding domain (ABS domain), giving it actin bundling activity. We show that spectrin repeats (SRs) 10-13 of N2G and the N-terminus of FHOD1 interacts with each other directly by biochemical assays with purified proteins. SiRNA against FHOD1 and overexpression of either FHOD1 or N2G interacting domains prevented LPA-stimulated nuclear movement in wounded monolayers of NIH3T3 fibroblasts, suggesting that the interaction between FHOD1 and N2G is required for nuclear movement and centrosome reorientation.

FHOD1 was required for TAN line formation, but was dispensable for the formation of dorsal actin cables and retrograde actin flow. By re-expressing an artificial construct containing the N2G-binding domain of FHOD1 and the actin-binding domain of α–actinin in FHOD1 depleted cells, we show that the FHOD1 ABS domain provides N2G with an additional contact to actin filaments required for nuclear movement. This study thus identifies FHOD1 as a new TAN line component and suggests that the interaction of FHOD1 with N2G may reinforce TAN lines so that they can resist the force necessary to move the nucleus. The above study identifies a new component in a pathway that actively moves the nucleus. We have far less knowledge about the mechanism that maintains the nucleus in position when it is not moving. For example, it is unknown whether the static nuclear positioning is an active process or simply an inactivation of mechanisms that actively move nuclei. To answer this question, I developed a novel method to artificially displace the nucleus in adherent cells by centrifugation and used this system to identify active mechanisms of homeostatic nuclear positioning.

By subjecting wounded monolayers of starved NIH3T3 fibroblast on coverslips to centrifugal force perpendicular to the wound, I find that nuclei are displaced towards the direction of centrifugal force, so that on one wound edge, the nuclei are in the cell rear while on the other, in the cell front. After returning centrifuged cells to the incubator, I used fixed and live cell recordings to show that the displaced nuclei actively re-center within one hour, although nuclei moving rearward did so faster than those moving forward. Treating centrifuged cells with cytoskeletal drugs, revealed an actin/myosin II-dependent rearward recentration and a microtubule (MT)/dynein-dependent forward recentration. I knocked down LINC complex components to test their involvement in these movements. N2G was required for both rearward and forward movement while SUN1 and SUN2 were required for forward and rearward movement, respectively. Overexpression of different N2G constructs in N2G-depleted cells showed that different regions of N2G were necessary for each direction of movement: N-terminal constructs rescued rearward nuclear recentration whereas C-terminal constructs rescued forward recentration.

Based on the minimal N2G construct that rescued forward (MT dependent) nuclear recentration, I identified a dynein and dynactin site in the C terminus of N2G. To test whether the homeostatic nuclear positioning mechanisms were active in uncentrifuged cells, I depleted cells of nesprin-2 and then re-expressed nesprin-2 constructs capable of interacting with actin, MTs or both cytoskeletal elements. Nuclei in nesprin-2-depleted cells were no longer maintained at the cell centroid and only re-expression of a construct that contained sites for interaction with both actin and MTs rescued this defect. Thus, both actin- and MT- interaction domains of N2G are required for homeostatic nuclear positioning. To test whether the actin and MT activities of N2G were important for cell migration, I depleted NIH3T3 fibroblasts of nesprin-2 and re-expressed N2G constructs capable of interaction with actin, MTs or both and tested these cells in single and collective cell migration assays. I found that only the MT-dependent activity of N2G is required for the directionality of single cell migration while both N- and C- terminal (actin- and MT- dependent) N2G are required for the velocity of collective cell migration.

These results show that different cytoskeletal linkages are used in different modes of cell migration. My thesis studies identify the first cytoplasmic factor required for TAN lines structure, establish a novel method to artificially displace the nucleus in adherent cells, and reveal different mechanisms of LINC complex coupling cytoskeletons during active and homeostatic nuclear positioning, as well as specific cytoskeleton-dependent contributions of nuclear envelope protein N2G during cell migration.