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Leukocytes patrol our bodies in search of pathogens and migrate to sites of injury in response to various stimuli. Rapid and directed leukocyte motility is therefore crucial to our immunity. The nucleus is the largest and stiffest cellular organelle and a mechanical obstacle for migration through constrictions. However, the nucleus is also essential for 3D cell migration. Here, we review the roles of the nucleus in leukocyte migration, focusing on how cells deform their nuclei to aid cell motility and the contributions of the nucleus to cell migration. We discuss the regulation of the nuclear biomechanics by the nuclear lamina and how it, together with the cytoskeleton, modulates the shapes of leukocyte nuclei. We then summarize the functions of nesprins and SUN proteins in leukocytes and discuss how forces are exerted on the nucleus. Finally, we examine the mechanical roles of the nucleus in cell migration, including its roles in regulating the direction of migration and path selection.

The linker of nucleoskeleton and cytoskeleton (LINC) complex, composed of outer and inner nuclear membrane Klarsicht, ANC-1, and Syne homology (KASH) and Sad1 and UNC-84 (SUN) proteins, respectively, connects the nucleus to cytoskeletal filaments and performs diverse functions including nuclear positioning, mechanotransduction, and meiotic chromosome movements. Recent studies have shed light on the source of this diversity by identifying factors associated with the complex that endow specific functions as well as those that differentially anchor the complex within the nucleus. Additional diversity may be provided by accessory factors that reorganize the complex into higher-ordered arrays. As core components of the LINC complex are associated with several diseases, understanding the role of accessory and anchoring proteins could provide insights into pathogenic mechanisms.

Myoblast migration is essential for muscle development and repair; however, the factors that contribute to the polarity of migrating myoblasts are relatively unknown. We find that randomly migrating C2C12 myoblasts orient their centrosomes in the direction of migration. Using wounded monolayers, we further show that centrosome orientation is stimulated by the serum factor lysophosphatidic acid (LPA) and involves the rearward movement of the nucleus while the centrosome is maintained at the cell centroid. The rate of nuclear movement correlated with that of actin retrograde flow and both cytochalasin D and blebbistatin prevented nuclear movement and centrosome orientation. Actin-dependent rearward nuclear movement in fibroblasts is mediated by assembly of nuclear membrane nesprin-2G and SUN2 LINC complexes into transmembrane actin-associated nuclear (TAN) lines anchored by A-type lamins and emerin. In C2C12 myoblasts, depletion of nesprin-2G, SUN2 or lamin A/C prevented nuclear movement and endogenous nesprin-2G and a chimeric GFP-mini-nesprin-2G formed TAN lines during nuclear movement. Depleting nesprin-2G strongly interfered with directed cell migration and reduced the efficiency of myoblast fusion into multinucleated myotubes. Our results show that nuclear movement contributes to centrosome orientation and polarity for efficient migration and fusion of myoblasts. Given that mutations in the genes encoding A-type lamins, nesprin-2 and SUN2 cause Emery-Dreifuss muscular dystrophy and related myopathies, our results have implications for understanding the mechanism of disease pathogenesis.

The rearward positioning of the nucleus is a characteristic feature of most migrating cells. Studies using wounded monolayers of fibroblasts and myoblasts have shown that this positioning is actively established before migration by the coupling of dorsal actin cables to the nuclear envelope through nesprin-2G and SUN2 linker of nucleoskeleton and cytoskeleton (LINC) complexes. During nuclear movement, these LINC complexes cluster along the actin cables to form adhesive structures known as transmembrane actin-associated nuclear (TAN) lines. Here we described experimental procedures for studying nuclear movement and TAN lines using wounded monolayers of fibroblasts and myoblasts, the acquisition of data using immunofluorescence microscopy and live-cell imaging, and methods for data analysis and quantification.

Emerin, a nuclear membrane protein, and myosin IIB contribute to nuclear movement by regulating the directionality of nuclear movement and dorsal actin cable flow. Emerin interacts with myosin IIB and is required for its perinuclear localization. The results show that the nuclear envelope actively organizes cytoplasmic polarity., In migrating fibroblasts, rearward movement of the nucleus orients the centrosome toward the leading edge. Nuclear movement results from coupling rearward-moving, dorsal actin cables to the nucleus by linear arrays of nesprin-2G and SUN2, termed transmembrane actin-associated nuclear (TAN) lines. A-type lamins anchor TAN lines, prompting us to test whether emerin, a nuclear membrane protein that interacts with lamins and TAN line proteins, contributes to nuclear movement. In fibroblasts depleted of emerin, nuclei moved nondirectionally or completely failed to move. Consistent with these nuclear movement defects, dorsal actin cable flow was nondirectional in cells lacking emerin. TAN lines formed normally in cells lacking emerin and were coordinated with the erratic nuclear movements, although in 20% of the cases, TAN lines slipped over immobile nuclei. Myosin II drives actin flow, and depletion of myosin IIB, but not myosin IIA, showed similar nondirectional nuclear movement and actin flow as in emerin-depleted cells. Myosin IIB specifically coimmunoprecipitated with emerin, and emerin depletion prevented myosin IIB localization near nuclei. These results show that emerin functions with myosin IIB to polarize actin flow and nuclear movement in fibroblasts, suggesting a novel function for the nuclear envelope in organizing directional actin flow and cytoplasmic polarity.