Transmembrane Actin-associated Nuclear Lines Research Articles

Ctdnep1 and Eps8L2 regulate dorsal actin cables for nuclear positioning during cell migration

SummaryCells actively position their nuclei within the cytoplasm for multiple cellular and physiological functions.1, 2, 3 Consequently, nuclear mispositioning is usually associated with cell dysfunction and disease, from muscular disorders to cancer metastasis.4, 5, 6, 7 Different cell types position their nuclei away from the leading edge during cell migration.8, 9, 10, 11 In migrating fibroblasts, nuclear positioning is driven by an actin retrograde flow originated at the leading edge that drives dorsal actin cables away from the leading edge. The dorsal actin cables connect to the nuclear envelope by the linker of nucleoskeleton and cytoskeleton (LINC) complex on transmembrane actin-associated nuclear (TAN) lines.12, 13, 14 Dorsal actin cables are required for the formation of TAN lines. How dorsal actin cables are organized to promote TAN lines formation is unknown. Here, we report a role for Ctdnep1/Dullard, a nuclear envelope phosphatase,15, 16, 17, 18, 19, 20, 21, 22 and the actin regulator Eps8L223, 24, 25 on nuclear positioning and cell migration. We demonstrate that Ctdnep1 and Eps8L2 directly interact, and this interaction is important for nuclear positioning and cell migration. We also show that Ctdnep1 and Eps8L2 are involved in the formation and thickness of dorsal actin cables required for TAN lines engagement during nuclear movement. We propose that Ctdnep1-Eps8L2 interaction regulates dorsal actin cables for nuclear movement during cell migration.

Methods to Measure Perinuclear Actin Dynamics During Nuclear Movement in Migrating Cells.

The nucleus is specifically positioned within a cell in diverse biological contexts. There are multiple connections between the nuclear envelope and the cytoskeleton and these connections are involved in nuclear positioning. During cell polarization prior to cell migration, nuclear envelope proteins bind to the actin cytoskeleton and get organized into linear arrays, known as transmembrane actin-associated nuclear (TAN) lines to move the nucleus away from the leading edge. Here we describe methods to study perinuclear actin dynamics, including measurement of the thickness of actin cables coupled to TAN lines, measurement of the number of perinuclear actin cables, and ablation of perinuclear actin cables. These methods are used to identify mechanisms of nuclear positioning.

TorsinA controls TAN line assembly and the retrograde flow of dorsal perinuclear actin cables during rearward nuclear movement.

The nucleus is positioned toward the rear of most migratory cells. In fibroblasts and myoblasts polarizing for migration, retrograde actin flow moves the nucleus rearward, resulting in the orientation of the centrosome in the direction of migration. In this study, we report that the nuclear envelope-localized AAA+ (ATPase associated with various cellular activities) torsinA (TA) and its activator, the inner nuclear membrane protein lamina-associated polypeptide 1 (LAP1), are required for rearward nuclear movement during centrosome orientation in migrating fibroblasts. Both TA and LAP1 contributed to the assembly of transmembrane actin-associated nuclear (TAN) lines, which couple the nucleus to dorsal perinuclear actin cables undergoing retrograde flow. In addition, TA localized to TAN lines and was necessary for the proper mobility of EGFP-mini-nesprin-2G, a functional TAN line reporter construct, within the nuclear envelope. Furthermore, TA and LAP1 were indispensable for the retrograde flow of dorsal perinuclear actin cables, supporting the recently proposed function for the nucleus in spatially organizing actin flow and cytoplasmic polarity. Collectively, these results identify TA as a key regulator of actin-dependent rearward nuclear movement during centrosome orientation.

Wound-Healing Assays to Study Mechanisms of Nuclear Movement in Fibroblasts and Myoblasts.

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.

Linker of nucleoskeleton and cytoskeleton (LINC) complex-mediated actin-dependent nuclear positioning orients centrosomes in migrating myoblasts

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.

Samp1 is a component of TAN lines and is required for nuclear movement

The position of the nucleus is regulated in different developmental stages and cellular events. During polarization, the nucleus moves away from the future leading edge and this movement is required for proper cell migration. Nuclear movement requires the LINC complex components nesprin-2G and SUN2, which form transmembrane actin-associated nuclear (TAN) lines at the nuclear envelope. Here we show that the nuclear envelope protein Samp1 (NET5) is involved in nuclear movement during fibroblast polarization and migration. Moreover, we demonstrate that Samp1 is a component of TAN lines that contain nesprin-2G and SUN2. Finally, Samp1 associates with SUN2 and lamin A/C, and the presence of Samp1 at the nuclear envelope requires lamin A/C. These results support a role for Samp1 in the association between the LINC complex and lamins during nuclear movement.

Nuclear cell biology

How does the nucleus move within the cell? How is the nucleus compartmentalized? How is nuclear size maintained? How is chromatin organized within the nucleus? These are a few of the questions related to nuclear cell biology addressed by our Minisymposium. Dan Starr led off with a description of how nuclei migrate in Caenorhabditis elegans. A KASH protein in the outer nuclear membrane recruits microtubule motors to the nucleus. Live imaging of nuclear migration and microtubules suggests that kinesin-1 provides the major force to move nuclei. To resolve large cellular roadblocks, dynein mediates backward movements or dramatic nuclear rolling events (Fridolfsson and Starr, 2010 ). G. W. Gant Luxton discussed mechanisms of nuclear migration in wounded cultured fibroblasts. Nesprin2G, SUN2, lamin, and actin cables associate in transmembrane actin-associated nuclear (TAN) lines to move nuclei (Luxton et al., 2010 ). The AAA+ ATPase TorsinA disease gene regulates the formation of TAN lines and nuclear migration. TorsinA knockdown reduced the localization and mobility of Nesprin2G and disrupted nuclear migration, suggesting that TorsinA regulates SUN-KASH interactions in the nuclear envelope. Levels of lamin-A/-C are known to increase during differentiation of embryonic stem cells (Constantinescu et al., 2006 ). Joe Swift described new mass spectrometry methods to accurately measure the ratios of lamin-A/-C to lamin-B isoforms, which had not been directly measured previously. The findings are just beginning to enhance our understanding of lamin changes throughout development and aging. Dan Levy described using Xenopus laevis and tropicalis as model organisms with similar DNA content but differently sized nuclei to understand nuclear scaling. Sperm nuclear assembly in tropicalis and laevis egg extracts mixed to different proportions directly correlated with nuclear size, suggesting size control by an egg cytoplasmic factor (Levy and Heald, 2010 ). Amounts of importin-α, Ntf2, and lamin were all involved in nuclear scaling. Future analyses will identify additional nuclear size factors and show how changes in size affect genome function. Jason Brickner described mechanisms of targeting a gene to the nuclear periphery in yeast. Genes were localized by LacO repeats and GFP-LacI. Mutational analyses revealed DNA elements that function as “DNA zip codes” to promote targeting to the nuclear periphery through association with the nuclear pore complex (Ahmed et al., 2010 ). A different DNA zip code controls posttranscriptional intranuclear targeting of the INO1 gene. This “memory” zip code is necessary and sufficient for H2A.Z incorporation and priming for reactivation (Light et al., 2010 ). Specific zip codes may also promote long-range interchromatin interactions between genomic regions sharing the same elements. Finally, Ana Pombo described how changes in chromatin positioning accompany gene activation, using the human uPA gene (Ferrai et al., 2010 ). Prior to induction, uPA alleles are found at the interior of their chromosome territory, associated with poised transcription factories characterized by RNA polymerase II phosphorylation on Serine5, but not Serine2. Gene activation with phorbol esters induced uPA gene repositioning out of its chromosome territory. Investigation of a looping requirement for transcriptional activation showed that gene looping is not required for activity upon induction, but that the internal chromosome territory position prior to activation is important for reinforcing the silent state. The Minisymposium on Nuclear Cell Biology covered the breadth of nuclear research, from events that control nuclear positioning within cells, to nuclear size control, to chromatin positioning and gene regulation. Future studies will focus on how nuclear position and size influence genome function, how nuclear envelope components might regulate the peripheral localization of individual loci, and how gene activity is regulated in different areas of the nucleus. We therefore look forward to future ASCB programs highlighting nuclear cell biology.

Lamin A variants that cause striated muscle disease are defective in anchoring transmembrane actin-associated nuclear lines for nuclear movement

Mutations in LMNA, which encodes A-type lamins, result in disparate diseases, known collectively as laminopathies, that affect distinct tissues, including striated muscle and adipose tissue. Lamins provide structural support for the nucleus and sites of attachment for chromatin, and defects in these functions may contribute to disease pathogenesis. Recent studies suggest that A-type lamins may facilitate connections between the nucleus and the cytoskeleton mediated by nuclear envelope nesprin and SUN proteins. In mammalian cells, however, interfering with A-type lamins does not affect the localization of these proteins. Here, we used centrosome orientation in fibroblasts, which requires separate nuclear and centrosome positioning pathways, as a model system to understand how LMNA mutations affect nucleus-cytoskeletal connections. We find that LMNA mutations causing striated muscle diseases block actin-dependent nuclear movement, whereas most that affect adipose tissue inhibit microtubule-dependent centrosome positioning. Genetic deletion or transient depletion of A-type lamins also blocked nuclear movement, showing that mutations affecting muscle exhibit the null phenotype. Lack of A-type lamins, or expression of variants that cause striated muscle disease, did not affect assembly of nesprin-2G and SUN2 into transmembrane actin-associated nuclear (TAN) lines that attach the nucleus to retrogradely moving actin cables. Nesprin-2G TAN lines were less stable, however, and slipped over the nucleus rather than moving with it, indicating that they were not anchored. Nesprin-2G TAN lines also slipped in SUN2-depleted cells. Our results establish A-type lamins as anchors for nesprin-2G-SUN2 TAN lines to allow productive movement and proper positioning of the nucleus by actin.