Reinforcing the LINC complex connection to actin filaments: the role of FHOD1 in TAN line formation and nuclear movement

Positioning the nucleus is critical for many cellular processes including cell division, migration and differentiation. The linker of nucleoskeleton and cytoskeleton (LINC) complex spans the inner and outer nuclear membranes and has emerged as a major factor in connecting the nucleus to the cytoskeleton for movement and positioning. Recently, we discovered that the diaphanous formin family member FHOD1 interacts with the LINC complex component nesprin-2 giant (nesprin-2G) and that this interaction plays essential roles in the formation of transmembrane actin-dependent nuclear (TAN) lines and nuclear movement during cell polarization in fibroblasts. We found that FHOD1 strengthens the connection between nesprin-2G and rearward moving dorsal actin cables by providing a second site of interaction between nesprin-2G and the actin cable. These results indicate that the LINC complex connection to the actin cytoskeleton can be enhanced by cytoplasmic factors and suggest a new model for TAN line formation. We discuss how the nesprin-2G-FHOD1 interaction may be regulated and its possible functional significance for development and disease.     

Emerging role for nuclear rotation and orientation in cell migration

Nucleus movement, positioning, and orientation is precisely specified and actively regulated within cells, and it plays a critical role in many cellular and developmental processes. Mutation of proteins that regulate the nucleus anchoring and movement lead to diverse pathologies, laminopathies in particular, suggesting that the nucleus correct positioning and movement is essential for proper cellular function. In motile cells that polarize toward the direction of migration, the nucleus undergoes controlled rotation promoting the alignment of the nucleus with the axis of migration. Such spatial organization of the cell appears to be optimal for the cell migration. Nuclear reorientation requires the cytoskeleton to be anchored to the nuclear envelope, which exerts pulling or pushing torque on the nucleus. Here we discuss the possible molecular mechanisms regulating the nuclear rotation and reorientation and the significance of this type of nuclear movement for cell migration.     

Emerging role for nuclear rotation and orientation in cell migration

Nucleus movement, positioning, and orientation is precisely specified and actively regulated within cells, and it plays a critical role in many cellular and developmental processes. Mutation of proteins that regulate the nucleus anchoring and movement lead to diverse pathologies, laminopathies in particular, suggesting that the nucleus correct positioning and movement is essential for proper cellular function. In motile cells that polarize toward the direction of migration, the nucleus undergoes controlled rotation promoting the alignment of the nucleus with the axis of migration. Such spatial organization of the cell appears to be optimal for the cell migration. Nuclear reorientation requires the cytoskeleton to be anchored to the nuclear envelope, which exerts pulling or pushing torque on the nucleus. Here we discuss the possible molecular mechanisms regulating the nuclear rotation and reorientation and the significance of this type of nuclear movement for cell migration.     

Myosin VI,an actin motor for membrane traffic and cell migration

The actin cytoskeleton and associated myosin motor proteins are essential for the transport and steady-state localization of vesicles and organelles and for the dynamic remodeling of the plasma membrane as well as for the maintenance of differentiated cell-surface structures. Myosin VI may be expected to have unique cellular functions, because it moves, unlike almost all other myosins, towards the minus end of actin filaments. Localization and functional studies indicate that myosin VI plays a role in a variety of different intracellular processes, such as endocytosis and secretion as well as cell migration. These diverse functions of myosin VI are mediated by interaction with a range of different binding partners .     

An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine

Genetic and biochemical evidence demonstrated that Eps8 is involved in the routing of signals from Ras to Rac. This is achieved through the formation of a tricomplex consisting of Eps8-E3b1-Sos-1, which is endowed with Rac guanine nucleotide exchange activity. The catalytic subunit of this complex is represented by Sos-1, a bifunctional molecule capable of catalyzing guanine nucleotide exchange on Ras and Rac. The mechanism by which Sos-1 activity is specifically directed toward Rac remains to be established. Here, by performing a structure-function analysis we show that the Eps8 output function resides in an effector region located within its COOH terminus. This effector region, when separated from the holoprotein, activates Rac and acts as a potent inducer of actin polymerization. In addition, it binds to Sos-1 and is able to induce Rac-specific, Sos-1-dependent guanine nucleotide exchange activity. Finally, the Eps8 effector region mediates a direct interaction of Eps8 with F-actin, dictating Eps8 cellular localization. We propose a model whereby the engagement of Eps8 in a tricomplex with E3b1 and Sos-1 facilitates the interaction of Eps8 with Sos-1 and the consequent activation of an Sos-1 Rac-specific catalytic ability. In this complex, determinants of Eps8 are responsible for the proper localization of the Rac-activating machine to sites of actin remodeling.     

Cathepsin E generates a sumoylated intracellular fragment of the cell adhesion molecule L1 to promote neuronal and Schwann cell migration as well as myelination

The cell adhesion molecule L1 regulates cellular responses in the developing and adult nervous system. Here, we show that stimulation of cultured mouse cerebellar neurons by a function‐triggering L1 antibody leads to cathepsin E‐mediated generation of a sumoylated 30 kDa L1 fragment (L1‐30) and to import of L1‐30 into the nucleus. Mutation of the sumoylation site at K₁₁₇₂ or the cathepsin E cleavage site at E₁₁₆₇ abolishes generation of L1‐30, while mutation of the nuclear localization signal at K₁₁₄₇ prevents nuclear import of L1‐30. Moreover, the aspartyl protease inhibitor pepstatin impairs the generation of L1‐30 and inhibits L1‐induced migration of cerebellar neurons and Schwann cells as well as L1‐dependent in vitro myelination on axons of dorsal root ganglion neurons by Schwann cells. L1‐stimulated migration of HEK293 cells expressing L1 with mutated cathepsin E cleavage site is diminished in comparison to migration of cells expressing non‐mutated L1. In addition, L1‐stimulated migration of HEK293 cells expressing non‐mutated L1 is also abolished upon knock‐down of cathepsin E expression and enhanced by over‐expression of cathepsin E. The findings of the present study indicate that generation and nuclear import of L1‐30 regulate neuronal and Schwann cell migration as well as myelination.

     

A role for CK2 upon interkinetic nuclear migration in the cell cycle of retinal progenitor cells

In developing retina, the nucleus of the elongated neuroepithelial cells undergoes interkinetic nuclear migration (INM), that is it migrates back and forth across the proliferative layer during the cell cycle. S-phase occurs at the basal side, while M-phase occurs at the apical margin of the retinal progenitors. G1 and G2-phases occur along the nuclear migration pathway. We tested whether this feature of the retinal cell cycle is controlled by CK2, which, among its many substrates, phosphorylates both molecular motors and cytoskeletal components. Double immunolabeling showed that CK2 is contained in BrdU-labeled retinal progenitors. INM was examined after pulse labeling the retina of newborn rats with BrdU, by plotting nuclear movement from basal to apical sides of the retinal progenitors during G2. The CK2 specific inhibitor 4,5,6,7-tetrabromobenzotriazole inhibited the activity of rat retinal CK2, and blocked nuclear movement proper in a dose-dependent way. No apoptosis was detected, and total numbers of BrdU-labeled nuclei remained constant following treatment. Immunohistochemistry showed that, following inhibition of CK2, the tubulin cytoskeleton is disorganized, with reduced acetylated and increased tyrosinated tubulin. This indicates a reduction in stable microtubules, with accumulation of free tubulin dimers. The results show that CK2 activity is required for INM in retinal progenitor cells.     

Protocadherin FAT1 binds Ena/VASP proteins and is necessary for actin dynamics and cell polarization

Cell migration requires integration of cellular processes resulting in cell polarization and actin dynamics. Previous work using tools of Drosophila genetics suggested that protocadherin fat serves in a pathway necessary for determining cell polarity in the plane of a tissue. Here we identify mammalian FAT1 as a proximal element of a signaling pathway that determines both cellular polarity in the plane of the monolayer and directed actin-dependent cell motility. FAT1 is localized to the leading edge of lamellipodia, filopodia, and microspike tips where FAT1 directly interacts with Ena/VASP proteins that regulate the actin polymerization complex. When targeted to mitochondrial outer leaflets, FAT1 cytoplasmic domain recruits components of the actin polymerization machinery sufficient to induce ectopic actin polymerization. In an epithelial cell wound model, FAT1 knockdown decreased recruitment of endogenous VASP to the leading edge and resulted in impairment of lamellipodial dynamics, failure of polarization, and an attenuation of cell migration. FAT1 may play an integrative role regulating cell migration by participating in Ena/VASP-dependent regulation of cytoskeletal dynamics at the leading edge and by transducing an Ena/VASP-independent polarity cue.     

NudC is required for interkinetic nuclear migration and neuronal migration during neocortical development

NudC is a highly conserved protein necessary for cytoplasmic dynein-mediated nuclear migration in Aspergillus nidulans. NudC interacts genetically with Aspergillus NudF and physically with its mammalian orthologue Lis1, which is crucial for nuclear and neuronal migration during brain development. To test for related roles for NudC, we performed in utero electroporation into embryonic rat brain of cDNAs encoding shRNAs as well as wild-type and mutant forms of NudC. We show here that NudC, like Lis1, is required for neuronal migration during neocorticogenesis and we identify a specific role in apical nuclear migration in radial glial progenitor cells. These results identify a novel neuronal migration gene with a specific role in interkinetic nuclear migration, consistent with cytoplasmic dynein regulation.     

Rapid and dynamic arginylation of the leading edge β‐actin is required for cell migration

β‐actin plays key roles in cell migration. Our previous work demonstrated that β‐actin in migratory non‐muscle cells is N‐terminally arginylated and that this arginylation is required for normal lamellipodia extension. Here, we examined the function of β‐actin arginylation in cell migration. We found that arginylated β‐actin is concentrated at the leading edge of lamellipodia and that this enrichment is abolished after serum starvation as well as in contact‐inhibited cells in confluent cultures, suggesting that arginylated β‐actin at the cell leading edge is coupled to active migration. Arginylated actin levels exhibit dynamic changes in response to cell stimuli, lowered after serum starvation and dramatically elevating within minutes after cell stimulation by readdition of serum or lysophosphatidic acid. These dynamic changes require active translation and are not seen in confluent contact‐inhibited cell cultures. Microinjection of arginylated actin antibodies into cells severely and specifically inhibits their migration rates. Together, these data strongly suggest that arginylation of β‐actin is a tightly regulated dynamic process that occurs at the leading edge of locomoting cells in response to stimuli and is integral to the signaling network that regulates cell migration.      

Ectodomain shedding of L1 adhesion molecule promotes cell migration by autocrine binding to integrins.

The L1 adhesion molecule plays an important role in axon guidance and cell migration in the nervous system. L1 is also expressed by many human carcinomas. In addition to cell surface expression, the L1 ectodomain can be released by a metalloproteinase, but the biological function of this process is unknown. Here we demonstrate that membrane-proximal cleavage of L1 can be detected in tumors and in the developing mouse brain. The shedding of L1 involved a disintegrin and metalloproteinase (ADAM)10, as transfection with dominant-negative ADAM10 completely abolishes L1 release. L1-transfected CHO cells (L1-CHO) showed enhanced haptotactic migration on fibronectin and laminin, which was blocked by antibodies to alpha v beta 5 and L1. Migration of L1-CHO cells, but not the basal migration of CHO cells, was blocked by a metalloproteinase inhibitor, indicating a role for L1 shedding in the migration process. CHO and metalloproteinase-inhibited L1-CHO cells were stimulated to migrate by soluble L1-Fc protein. The induction of migration was blocked by alpha v beta 5-specific antibodies and required Arg-Gly-Asp sites in L1. A 150-kD L1 fragment released by plasmin could also stimulate CHO cell migration. We propose that ectodomain-released L1 promotes migration by autocrine/paracrine stimulation via alpha v beta 5. This regulatory loop could be relevant for migratory processes under physiological and pathophysiological conditions.     

CYLD coordinates with EB1 to regulate microtubule dynamics and cell migration

Cylindromatosis (CYLD), a deubiquitinase involved in inflammation and tumorigenesis via the modulation of cell signaling, has recently been identified as a critical regulator of microtubule dynamics. CYLD has also been shown to stimulate cell migration and thereby contribute to normal physiological processes. However, it remains elusive how the regulation of microtubule dynamic properties by CYLD is connected to its role in mediating cell migration. In this study, we performed yeast 2-hybrid screening with CYLD as bait and identified 7 CYLD-interacting proteins, including end-binding protein 1 (EB1). The CYLD–EB1 interaction was confirmed both in cells and in vitro, and these 2 proteins colocalized at the plus ends of microtubules. Interestingly, the association of CYLD with EB1 was significantly increased upon the stimulation of cell migration. CYLD coordinated with EB1 to orchestrate tail retraction, centrosome reorientation, and leading-edge microtubule stabilization in migratory cells. In addition, CYLD acted in concert with EB1 to regulate microtubule assembly in vitro, microtubule nucleation at the centrosome, and microtubule growth at the cell periphery. These data provide mechanistic insights into the actions of CYLD in the regulation of microtubule dynamics and cell migration. These findings also support the notion that coordinated actions of microtubule-binding proteins are critical for microtubule-mediated cellular events.     

Mammalian CAP (Cyclase-associated protein) in the world of cell migration: Roles in actin filament dynamics and beyond

Cell migration is essential for a variety of fundamental biological processes such as embryonic development, wound healing, and immune response. Aberrant cell migration also underlies pathological conditions such as cancer metastasis, in which morphological transformation promotes spreading of cancer to new sites. Cell migration is driven by actin dynamics, which is the repeated cycling of monomeric actin (G-actin) into and out of filamentous actin (F-actin). CAP (Cyclase-associated protein, also called Srv2) is a conserved actin-regulatory protein, which is implicated in cell motility and the invasiveness of human cancers. It cooperates with another actin regulatory protein, cofilin, to accelerate actin dynamics. Hence, knockdown of CAP1 slows down actin filament turnover, which in most cells leads to reduced cell motility. However, depletion of CAP1 in HeLa cells, while causing reduction in dynamics, actually led to increased cell motility. The increases in motility are likely through activation of cell adhesion signals through an inside-out signaling. The potential to activate adhesion signaling competes with the negative effect of CAP1 depletion on actin dynamics, which would reduce cell migration. In this commentary, we provide a brief overview of the roles of mammalian CAP1 in cell migration, and highlight a likely mechanism underlying the activation of cell adhesion signaling and elevated motility caused by depletion of CAP1.     

PTPRN2 and PLCβ1 promote metastatic breast cancer cell migration through PI(4,5)P2‐dependent actin remodeling

Altered abundance of phosphatidyl inositides (PIs) is a feature of cancer. Various PIs mark the identity of diverse membranes in normal and malignant cells. Phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P₂) resides predominantly in the plasma membrane, where it regulates cellular processes by recruiting, activating, or inhibiting proteins at the plasma membrane. We find that PTPRN2 and PLCβ1 enzymatically reduce plasma membrane PI(4,5)P₂ levels in metastatic breast cancer cells through two independent mechanisms. These genes are upregulated in highly metastatic breast cancer cells, and their increased expression associates with human metastatic relapse. Reduction in plasma membrane PI(4,5)P₂ abundance by these enzymes releases the PI(4,5)P₂‐binding protein cofilin from its inactive membrane‐associated state into the cytoplasm where it mediates actin turnover dynamics, thereby enhancing cellular migration and metastatic capacity. Our findings reveal an enzymatic network that regulates metastatic cell migration through lipid‐dependent sequestration of an actin‐remodeling factor.     

Structures of FHOD1-Nesprin1/2 complexes reveal alternate binding modes for the FH3 domain of formins

1. Download : Download high-res image (190KB)
2. Download : Download full-size image

     

Interleukin-8 induces the endothelial cell migration through the activation of phosphoinositide 3-kinase-Rac1/RhoA pathway

Endothelial cell migration is essential for tumor angiogenesis, and interleukin-8 (IL-8) has been shown to play an important role in tumor growth, angiogenesis, and metastasis. This study aimed to investigate the molecular mechanism of IL-8 induced endothelial cell migration. Our results indicated that IL-8 induced a rapid rearrangement of the actin cytoskeleton in EA.Hy926 cells, generating extensions resembling membrane ruffling and stress fibers. These processes required parallel upregulation of the small GTPases Rac1 and RhoA. Moreover, we demonstrated that IL-8 activated PI3K following the same kinetics observed from IL-8 induction of cytoskeletal rearrangement, suggesting the participation of PI3K in these processes. Taken together, our study demonstrates that PI3K-Rac1/RhoA signaling pathway plays a vital role in IL-8 induced endothelial cell migration, and provides new insight into the molecular mechanisms by which IL-8 contributes to tumor angiogenesis and metastasis.     

Distinct functions for ERK1 and ERK2 in cell migration processes during zebrafish gastrulation

The MAPKs are key regulatory signaling molecules in many cellular processes. Here we define differential functions for ERK1 and ERK2 MAPKs in zebrafish embryogenesis. Morpholino knockdown of ERK1 and ERK2 resulted in cell migration defects during gastrulation, which could be rescued by co-injection of the corresponding mRNA. Strikingly, Erk2 mRNA cross-rescued ERK1 knockdown, but erk1 mRNA was unable to compensate for ERK2 knockdown. Cell-tracing experiments revealed a convergence defect for ERK1 morphants without a severe posterior-extension defect, whereas ERK2 morphants showed a more severe reduction in anterior-posterior extension. These defects were primary changes in gastrulation cell movements and not caused by altered cell fate specification. Saturating knockdown conditions showed that the absence of FGF-mediated dual-phosphorylated ERK2 from the blastula margin blocked initiation of epiboly, actin and tubulin cytoskeleton reorganization processes and further arrested embryogenesis, whereas ERK1 knockdown had only a mild effect on epiboly progression. Together, our data define distinct roles for ERK1 and ERK2 in developmental cell migration processes during zebrafish embryogenesis.