Directional cell migration is a fundamental process in all organisms that is stringently regulated during tissue development, chemotaxis and wound healing. Migrating cells have a polarized morphology ...with an asymmetrical distribution of signaling molecules and the cytoskeleton. Microtubules are indispensable for the directional migration of certain cells. Recent studies have shown that Rho family GTPases, which are key regulators of cell migration, affect microtubules, in addition to the actin cytoskeleton and adhesion. Rho family GTPases capture and stabilize microtubules through their effectors at the cell cortex, leading to a polarized microtubule array; in turn, microtubules modulate the activities of Rho family GTPases. In this article, we discuss how a polarized microtubule array is established and how microtubules facilitate cell migration.
Members of the Rho family of small Ras-like GTPases-including RhoA,
-B, and -C, Rac1 and -2, and Cdc42-exhibit guanine nucleotide-binding
activity and function as molecular switches, cycling between ...an inactive
GDP-bound state and an active GTP-bound state. The Rho family GTPases
participate in regulation of the actin cytoskeleton and cell adhesion through
specific targets. Identification and characterization of these targets have
begun to clarify how the Rho family GTPases act to regulate cytoskeletal
structure and cell-cell and cell-substratum contacts in mammalian cells. The
Rho family GTPases are also involved in regulation of smooth muscle
contraction, cell morphology, cell motility, neurite retraction, and
cytokinesis. However, the molecular mechanisms by which the Rho family GTPases
participate in the regulation of such processes are not well established.
Regulated increase in the formation of microtubule arrays is thought to be important for axonal growth. Collapsin response mediator protein-2 (CRMP-2) is a mammalian homologue of UNC-33, mutations in ...which result in abnormal axon termination. We recently demonstrated that CRMP-2 is critical for axonal differentiation. Here, we identify two activities of CRMP-2: tubulin-heterodimer binding and the promotion of microtubule assembly. CRMP-2 bound tubulin dimers with higher affinity than it bound microtubules. Association of CRMP-2 with microtubules was enhanced by tubulin polymerization in the presence of CRMP-2. The binding property of CRMP-2 with tubulin was apparently distinct from that of Tau, which preferentially bound microtubules. In neurons, overexpression of CRMP-2 promoted axonal growth and branching. A mutant of CRMP-2, lacking the region responsible for microtubule assembly, inhibited axonal growth and branching in a dominant-negative manner. Taken together, our results suggest that CRMP-2 regulates axonal growth and branching as a partner of the tubulin heterodimer, in a different fashion from traditional MAPs.
Disrupted‐in‐schizophrenia 1 (Disc1) is a key molecular driver for the biology of mental diseases. In order to investigate its role in brain function, we previously generated mice lacking exons 2 and ...3 of Disc1 on a C57BL/6J genetic background (Disc1Δ2‐3/Δ2‐3 mice), which have a deficiency of the full‐length Disc1 protein. In the present study, we examined the role of Disc1 in cognitive function using a touchscreen‐based visual discrimination (VD) task in which mice had to discriminate 1 of 2 stimuli simultaneously displayed on the screen and received a liquid reward. Disc1Δ2‐3/Δ2‐3 mice showed impaired performance in the VD task, and this was mainly attributed to the perseverative response being significantly stronger than that in wild‐type (WT) mice. Furthermore, the numbers of marbles buried in the marble burying test and nestlets shredded in the nestlet shredding test by Disc1Δ2‐3/Δ2‐3 mice were significantly higher than those by WT mice, suggesting perseverative/compulsive behaviors by Disc1Δ2‐3/Δ2‐3 mice. A treatment with clozapine ameliorated behavioral deficits in the VD and marble burying tasks. c‐Fos expression was significantly stronger in the dorsomedial striatum (DMS), but not the dorsolateral striatum (DLS) after the first VD session in Disc1Δ2‐3/Δ2‐3 mice than in WT mice. The treatment of mice that had previously expressed hM3Dq in the DMS with clozapine‐N‐oxide (CNO) impaired performance in the VD task. These results suggest that cognitive impairments accompanied by perseverative/compulsive behaviors in Disc1Δ2‐3/Δ2‐3 mice are associated with hyperactivity of the DMS.
By employing behavioral, pharmacological, brain‐region mapping and neuronal manipulation methods, we found that the loss of the Disrupted‐in‐schizophrenia‐1 (Disc1) protein resulted in a decreased ability in mice to learn the response‐outcome association, which was mainly due to hyperactivity observed in the dorsomedial striatum (DMS) of these mice.
Axon growth during neural development is highly dependent on both cytoskeletal re-organization and polarized membrane trafficking. Previously, we demonstrated that collapsin response mediator ...protein-2 (CRMP-2) is critical for specifying axon/dendrite fate and axon growth in cultured hippocampal neurons, possibly by interacting with tubulin heterodimers and promoting microtubule assembly. Here, we identify Numb as a CRMP-2-interacting protein. Numb has been shown to interact with α-adaptin and to be involved in endocytosis. We found that Numb was associated with L1, a neuronal cell adhesion molecule that is endocytosed and recycled at the growth cone, where CRMP-2 and Numb were colocalized. Furthermore, expression of dominant-negative CRMP-2 mutants or knockdown of CRMP-2 message with small-interfering (si) RNA inhibited endocytosis of L1 at axonal growth cones and suppressed axon growth. These results suggest that in addition to regulating microtubule assembly, CRMP-2 is involved in polarized Numb-mediated endocytosis of proteins such as L1.
The small GTP binding protein Rho is implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid to form stress fibers and focal contacts. Here we have purified a ...Rho‐interacting protein with a molecular mass of approximately 164 kDa (p164) from bovine brain. This protein bound to GTPgammaS (a non‐hydrolyzable GTP analog).RhoA but not to GDP.RhoA or GTPgammaS.RhoA with a mutation in the effector domain (RhoAA37).p164 had a kinase activity which was specifically stimulated by GTPgammaS.RhoA. We obtained the cDNA encoding p164 on the basis of its partial amino acid sequences and named it Rho‐associated kinase (Rho‐kinase). Rho‐kinase has a catalytic domain in the N‐terminal portion, a coiled coil domain in the middle portion and a zinc finger‐like motif in the C‐terminal portion. The catalytic domain shares 72% sequence homology with that of myotonic dystrophy kinase and the coiled coil domain contains a Rho‐interacting interface. When COS7 cells were cotransfected with Rho‐kinase and activated RhoA, some Rho‐kinase was recruited to membranes. Thus it is likely that Rho‐kinase is a putative target serine/threonine kinase for Rho and serves as a mediator of the Rho‐dependent signaling pathway.
Endothelial differentiation gene (Edg) proteins are G-protein-coupled receptors activated by lysophospholipid mediators: sphingosine-1-phosphate (S1P) or lysophosphatidic acid. We show that in the ...CNS, expression of Edg8/S1P5, a high-affinity S1P receptor, is restricted to oligodendrocytes and expressed throughout development from the immature stages to the mature myelin-forming cell. S1P activation of Edg8/S1P5 on O4-positive pre-oligodendrocytes induced process retraction via a Rho kinase/collapsin response-mediated protein signaling pathway, whereas no retraction was elicited by S1P on these cells derived from Edg8/S1P5-deficient mice. Edg8/S1P5-mediated process retraction was restricted to immature cells and was no longer observed at later developmental stages. In contrast, S1P activation promoted the survival of mature oligodendrocytes but not of pre-oligodendrocytes. The S1P-induced survival of mature oligodendrocytes was mediated through a pertussis toxin-sensitive, Akt-dependent pathway. Our data demonstrate that Edg8/S1P5 activation on oligodendroglial cells modulates two distinct functional pathways mediating either process retraction or cell survival and that these effects depend on the developmental stage of the cell.
Migrating cells extend protrusions to establish new adhesion sites at their leading edges. One of the driving forces for cell migration is the directional trafficking of cell-adhesion molecules such ...as integrins. Here, we show that the endocytic adaptor protein Numb is an important component of the machinery for directional integrin trafficking in migrating cells. Numb binds to integrin-βs and localizes to clathrin-coated structures (CCSs) at the substratum-facing surface of the leading edge. Numb inhibition by RNAi impairs both integrin endocytosis and cell migration toward integrin substrates. Numb is regulated by phosphorylation since the protein is released from CCSs and no longer binds integrins when phosphorylated by atypical protein kinase C (aPKC). Because Numb interacts with the aPKC binding partner PAR-3, we propose a model in which polarized Numb phosphorylation contributes to cell migration by directing integrin endocytosis to the leading edge.
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