Eric S. Folker

Linear Arrays of Nuclear Envelope Proteins Harness Retrograde Actin Flow for Nuclear Movement

Line Up for Movement The nuclei of animal cells can move to specific locations and help to polarize migrating and differentiating cells. Luxton et al. (p. 956; see the Perspective by Starr) found that linear arrays of nuclear membrane proteins assembled on, and moved with, actin cables toward the rear of the cell during nuclear movement in polarizing fibroblasts. Interfering with the components of these linear arrays prevented nuclear movement and centrosome reorientation. Thus, nuclear membrane proteins assemble into actin-dependent arrays during force transduction. An actin-dependent mechanism is involved in moving nuclei so that they are properly positioned for cell migration. Nuclei move to specific locations to polarize migrating and differentiating cells. Many nuclear movements are microtubule-dependent. However, nuclear movement to reorient the centrosome in migrating fibroblasts occurs through an unknown actin-dependent mechanism. We found that linear arrays of outer (nesprin2G) and inner (SUN2) nuclear membrane proteins assembled on and moved with retrogradely moving dorsal actin cables during nuclear movement in polarizing fibroblasts. Inhibition of nesprin2G, SUN2, or actin prevented nuclear movement and centrosome reorientation. The coupling of actin cables to the nuclear membrane for nuclear movement via specific membrane proteins indicates that, like plasma membrane integrins, nuclear membrane proteins assemble into actin-dependent arrays for force transduction.

MAP and kinesin-dependent nuclear positioning is required for skeletal muscle function

The basic unit of skeletal muscle in all metazoans is the multinucleate myofibre, within which individual nuclei are regularly positioned. The molecular machinery responsible for myonuclear positioning is not known. Improperly positioned nuclei are a hallmark of numerous diseases of muscle, including centronuclear myopathies, but it is unclear whether correct nuclear positioning is necessary for muscle function. Here we identify the microtubule-associated protein ensconsin (Ens)/microtubule-associated protein 7 (MAP7) and kinesin heavy chain (Khc)/Kif5b as essential, evolutionarily conserved regulators of myonuclear positioning in Drosophila and cultured mammalian myotubes. We find that these proteins interact physically and that expression of the Kif5b motor domain fused to the MAP7 microtubule-binding domain rescues nuclear positioning defects in MAP7-depleted cells. This suggests that MAP7 links Kif5b to the microtubule cytoskeleton to promote nuclear positioning. Finally, we show that myonuclear positioning is physiologically important. Drosophila ens mutant larvae have decreased locomotion and incorrect myonuclear positioning, and these phenotypes are rescued by muscle-specific expression of Ens. We conclude that improper nuclear positioning contributes to muscle dysfunction in a cell-autonomous fashion.

Dynamics and molecular interactions of linker of nucleoskeleton and cytoskeleton (LINC) complex proteins

The linker of nucleoskeleton and cytoskeleton (LINC) complex is situated in the nuclear envelope and forms a connection between the lamina and cytoskeletal elements. Sun1, Sun2 and nesprin-2 are important components of the LINC complex. We expressed these proteins fused to green fluorescent protein in embryonic fibroblasts and studied their diffusional mobilities using fluorescence recovery after photobleaching. We show that they all are more mobile in embryonic fibroblasts from mice lacking A-type lamins than in cells from wild-type mice. Knockdown of Sun2 also increased the mobility of a short, chimeric form of nesprin-2 giant (mini-nesprin-2G), whereas the lack of emerin did not affect the mobility of Sun1, Sun2 or mini-nesprin-2G. Fluorescence resonance energy transfer experiments showed Sun1 to be more closely associated with lamin A than is Sun2. Sun1 and Sun2 had similar affinity for the nesprin-2 KASH domain in plasmon surface resonance (Biacore) experiments. This affinity was ten times higher than that previously reported between nesprin-2 and actin. Deletion of the actin-binding domain had no effect on mini-nesprin-2G mobility. Our data support a model in which A-type lamins and Sun2 anchor nesprin-2 in the outer nuclear membrane, whereas emerin, Sun1 and actin are dispensable for this anchoring.

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.Nuclear position is actively controlled and can be adjusted according to the needs of a cell by nuclear movement. Microtubules mediate the majority of nuclear movements studied to date, although examples of nuclear movements mediated by the actin cytoskeleton have been described. One such actin-dependent nuclear movement occurs during centrosome orientation in fibroblasts polarizing for migration. Here, the centrosome is maintained at the cell center while the nucleus is moved to the cell rear by actin retrograde flow thus positioning the centrosome between the nucleus and the leading edge of the cell. We have explored the molecular mechanism for actin dependent movement of the nucleus during centrosome centration. We found that a novel linear array of nuclear envelope membrane proteins composed of nesprin-2G and SUN2, called transmembrane actin-associated nuclear (TAN) lines, couple the nucleus to moving actin cables resulting in the nucleus being positioned toward the cell rear. TAN lines are anchored by A-type lamins and this allows the forces generated by the actin cytoskeleton to be transmitted across the nuclear envelope to move the nucleus. Here we review the data supporting this mechanism for nuclear movement, discuss questions remaining to be addressed and consider how this new mechanism of nuclear movement may shed light on human disease.

TAN lines: A novel nuclear envelope structure involved in nuclear positioning

Nuclear position is actively controlled and can be adjusted according to the needs of a cell by nuclear movement. Microtubules mediate the majority of nuclear movements studied to date, although examples of nuclear movements mediated by the actin cytoskeleton have been described. One such actin-dependent nuclear movement occurs during centrosome orientation in fibroblasts polarizing for migration. Here, the centrosome is maintained at the cell center while the nucleus is moved to the cell rear by actin retrograde flow thus positioning the centrosome between the nucleus and the leading edge of the cell. We have explored the molecular mechanism for actin dependent movement of the nucleus during centrosome centration. We found that a novel linear array of nuclear envelope membrane proteins composed of nesprin-2G and SUN2, called transmembrane actin-associated nuclear (TAN) lines, couple the nucleus to moving actin cables resulting in the nucleus being positioned toward the cell rear. TAN lines are anchored by A-type lamins and this allows the forces generated by the actin cytoskeleton to be transmitted across the nuclear envelope to move the nucleus. Here we review the data supporting this mechanism for nuclear movement, discuss questions remaining to be addressed and consider how this new mechanism of nuclear movement may shed light on human disease.

Nuclear positioning in muscle development and disease

Muscle disease as a group is characterized by muscle weakness, muscle loss, and impaired muscle function. Although the phenotype is the same, the underlying cellular pathologies, and the molecular causes of these pathologies, are diverse. One common feature of many muscle disorders is the mispositioning of myonuclei. In unaffected individuals, myonuclei are spaced throughout the periphery of the muscle fiber such that the distance between nuclei is maximized. However, in diseased muscles, the nuclei are often clustered within the center of the muscle cell. Although this phenotype has been acknowledged for several decades, it is often ignored as a contributor to muscle weakness. Rather, these nuclei are taken only as a sign of muscle repair. Here we review the evidence that mispositioned myonuclei are not merely a symptom of muscle disease but also a cause. Additionally, we review the working models for how myonuclei move from two different perspectives: from that of the nuclei and from that of the cytoskeleton. We further compare and contrast these mechanisms with the mechanisms of nuclear movement in other cell types both to draw general themes for nuclear movement and to identify muscle-specific considerations. Finally, we focus on factors that can be linked to muscle disease and find that genes that regulate myonuclear movement and positioning have been linked to muscular dystrophy. Although the cause-effect relationship is largely speculative, recent data indicate that the position of nuclei should no longer be considered only a means to diagnose muscle disease.

Emerin organizes actin flow for nuclear movement and centrosome orientation in migrating fibroblasts

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.

Muscle length and myonuclear position are independently regulated by distinct Dynein pathways

Various muscle diseases present with aberrant muscle cell morphologies characterized by smaller myofibers with mispositioned nuclei. The mechanisms that normally control these processes, whether they are linked, and their contribution to muscle weakness in disease, are not known. We examined the role of Dynein and Dynein-interacting proteins during Drosophila muscle development and found that several factors, including Dynein heavy chain, Dynein light chain and Partner of inscuteable, contribute to the regulation of both muscle length and myonuclear positioning. However, Lis1 contributes only to Dynein-dependent muscle length determination, whereas CLIP-190 and Glued contribute only to Dynein-dependent myonuclear positioning. Mechanistically, microtubule density at muscle poles is decreased in CLIP-190 mutants, suggesting that microtubule-cortex interactions facilitate myonuclear positioning. In Lis1 mutants, Dynein hyperaccumulates at the muscle poles with a sharper localization pattern, suggesting that retrograde trafficking contributes to muscle length. Both Lis1 and CLIP-190 act downstream of Dynein accumulation at the cortex, suggesting that they specify Dynein function within a single location. Finally, defects in muscle length or myonuclear positioning correlate with impaired muscle function in vivo, suggesting that both processes are essential for muscle function.

Interactions between EB1 and Microtubules: DRAMATIC EFFECT OF AFFINITY TAGS AND EVIDENCE FOR COOPERATIVE BEHAVIOR*

Plus end tracking proteins (+TIPs) are a unique group of microtubule binding proteins that dynamically track microtubule (MT) plus ends. EB1 is a highly conserved +TIP with a fundamental role in MT dynamics, but it remains poorly understood in part because reported EB1 activities have differed considerably. One reason for this inconsistency could be the variable presence of affinity tags used for EB1 purification. To address this question and establish the activity of native EB1, we have measured the MT binding and tubulin polymerization activities of untagged EB1 and EB1 fragments and compared them with those of His-tagged EB1 proteins. We found that N-terminal His tags directly influence the interaction between EB1 and MTs, significantly increasing both affinity and activity, and that small amounts of His-tagged proteins act synergistically with larger amounts of untagged proteins. Moreover, the binding ratio between EB1 and tubulin can exceed 1:1, and EB1-MT binding curves do not fit simple binding models. These observations demonstrate that EB1 binding is not limited to the MT seam, and they suggest that EB1 binds cooperatively to MTs. Finally, we found that removal of tubulin C-terminal tails significantly reduces EB1 binding, indicating that EB1-tubulin interactions are mediated in part by the same tubulin acidic tails utilized by other MAPs. These binding relationships are important for helping to elucidate the complex of proteins at the MT tip.

Translocating myonuclei have distinct leading and lagging edges that require Kinesin and Dynein

Nuclei are precisely positioned within all cells, and mispositioned nuclei are a hallmark of many muscle diseases. Myonuclear positioning is dependent on Kinesin and Dynein, but interactions between these motor proteins and their mechanisms of action are unclear. We find that in developing Drosophila muscles, Dynein and Kinesin work together to move nuclei in a single direction by two separate mechanisms that are spatially segregated. First, the two motors work together in a sequential pathway that acts from the cell cortex at the muscle poles. This mechanism requires Kinesin-dependent localization of Dynein to cell cortex near the muscle pole. From this location Dynein can pull microtubule minus-ends and the attached myonuclei toward the muscle pole. Second, the motors exert forces directly on individual nuclei independently of the cortical pathway. However, the activities of the two motors on the nucleus are polarized relative to the direction of myonuclear translocation: Kinesin acts at the leading edge of the nucleus, whereas Dynein acts at the lagging edge of the nucleus. Consistent with the activities of Kinesin and Dynein being polarized on the nucleus, nuclei rarely change direction, and those that do, reorient to maintain the same leading edge. Conversely, nuclei in both Kinesin and Dynein mutant embryos change direction more often and do not maintain the same leading edge when changing directions. These data implicate Kinesin and Dynein in two distinct and independently regulated mechanisms of moving myonuclei, which together maximize the ability of myonuclei to achieve their proper localizations within the constraints imposed by embryonic development.