Helen K. Matthews

Contact inhibition of locomotion in vivo controls neural crest directional migration

Contact inhibition of locomotion was discovered by Abercrombie more than 50 years ago and describes the behaviour of fibroblast cells confronting each other in vitro, where they retract their protrusions and change direction on contact. Its failure was suggested to contribute to malignant invasion. However, the molecular basis of contact inhibition of locomotion and whether it also occurs in vivo are still unknown. Here we show that neural crest cells, a highly migratory and multipotent embryonic cell population, whose behaviour has been likened to malignant invasion, demonstrate contact inhibition of locomotion both in vivo and in vitro, and that this accounts for their directional migration. When two migrating neural crest cells meet, they stop, collapse their protrusions and change direction. In contrast, when a neural crest cell meets another cell type, it fails to display contact inhibition of locomotion; instead, it invades the other tissue, in the same manner as metastatic cancer cells. We show that inhibition of non-canonical Wnt signalling abolishes both contact inhibition of locomotion and the directionality of neural crest migration. Wnt-signalling members localize at the site of cell contact, leading to activation of RhoA in this region. These results provide the first example of contact inhibition of locomotion in vivo, provide an explanation for coherent directional migration of groups of cells and establish a previously unknown role for non-canonical Wnt signalling.

Directional migration of neural crest cells in vivo is regulated by Syndecan-4/Rac1 and non-canonical Wnt signaling/RhoA

Directed cell migration is crucial for development, but most of our current knowledge is derived from in vitro studies. We analyzed how neural crest (NC) cells migrate in the direction of their target during embryonic development. We show that the proteoglycan Syndecan-4 (Syn4) is expressed in the migrating neural crest of Xenopus and zebrafish embryos. Loss-of-function studies using an antisense morpholino against syn4 show that this molecule is required for NC migration, but not for NC induction. Inhibition of Syn4 does not affect the velocity of cell migration, but significantly reduces the directional migration of NC cells. Furthermore, we show that Syn4 and PCP signaling control the directional migration of NC cells by regulating the direction in which the cell protrusions are generated during migration. Finally, we perform FRET analysis of Cdc42, Rac and RhoA in vitro and in vivo after interfering with Syn4 and PCP signaling. This is the first time that FRET analysis of small GTPases has been performed in vivo. Our results show that Syn4 inhibits Rac activity, whereas PCP signaling promotes RhoA activity. In addition, we show that RhoA inhibits Rac in NC cells. We present a model in which Syn4 and PCP control directional NC migration by, at least in part, regulating membrane protrusions through the regulation of small GTPase activities.

Changes in Ect2 localization couple actomyosin-dependent cell shape changes to mitotic progression.

Summary As they enter mitosis, animal cells undergo profound actin-dependent changes in shape to become round. Here we identify the Cdk1 substrate, Ect2, as a central regulator of mitotic rounding, thus uncovering a link between the cell-cycle machinery that drives mitotic entry and its accompanying actin remodeling. Ect2 is a RhoGEF that plays a well-established role in formation of the actomyosin contractile ring at mitotic exit, through the local activation of RhoA. We find that Ect2 first becomes active in prophase, when it is exported from the nucleus into the cytoplasm, activating RhoA to induce the formation of a mechanically stiff and rounded metaphase cortex. Then, at anaphase, binding to RacGAP1 at the spindle midzone repositions Ect2 to induce local actomyosin ring formation. Ect2 localization therefore defines the stage-specific changes in actin cortex organization critical for accurate cell division.

Exploring the function of cell shape and size during mitosis.

Dividing cells almost always adopt a spherical shape. This is true of most eukaryotic cells lacking a rigid cell wall and is observed in tissue culture and single-celled organisms, as well as in cells dividing inside tissues. While the mechanisms underlying this shape change are now well described, the functional importance of the spherical mitotic cell for the success of cell division has been thus far scarcely addressed. Here we discuss how mitotic rounding contributes to spindle assembly and positioning, as well as the potential consequences of abnormal mitotic cell shape and size on chromosome segregation, tissue growth, and cancer.

Directional cell migration in vivo: Wnt at the crest.

Directional cell migration is essential for almost all organisms during embryonic development, in adult life and contributes to pathological conditions. This is particularly critical during embryogenesis where it is essential that cells end up in their correct, precise locations in order to build a normal embryo. Many cells have solved this problem by following a gradient of a chemoattractant usually secreted by their target tissues. Our recent research has found an alternative, complimentary, mechanism where intracellular signals are able to generate cell polarity and directional migration in absence of any external chemoattactant. We used neural crest cells to study cell migration in vivo, by performing live imagining of the neural crest cell migrating during embryo development. We show that the Planar Cell Polarity (PCP) or non-canonical Wnt signaling pathway interacts with the proteoglycan syndecan-4 to control the direction in which cell protrusions are generated, and in consequence, the direction of migration. By analyzing the activity of the small GTPases using in vivo FRET imaging we showed that PCP signaling activates RhoA, while syndecan-4 inhibits Rac, both at the back of the neural crest cell. Here we discuss a model where these signals are integrated to generate directional migration in vivo.

Wnt11r is required for cranial neural crest migration

wnt11r is a recently identified member of the Wnt family of genes, which has been proposed to be the true Xenopus homologue to the mammalian wnt11 gene. In this study we have examined the role of wnt11r on neural crest development. Expression analysis of wnt11r and comparison with the neural crest marker snail2 and the noncanonical Wnt, wnt11, shows wnt11r is expressed at the medial or neural plate side of the neural crest while wnt11 is expressed at the lateral or epidermal side. Injection of wnt11r morpholino leads to strong inhibition of neural crest migration with no effect on neural crest induction or maintenance. This effect can be rescued by co‐injection of Wnt11r but not by Wnt11 mRNA, demonstrating the specificity of the loss of function treatment. Finally, neural crest graft experiments show that wnt11r is required in a non–cell‐autonomous manner to control neural crest migration. Developmental Dynamics 237:3404–3409, 2008.

The metastatic cancer cell cortex: An adaptation to enhance robust cell division in novel environments?

To metastasize, cancer cells must be able to complete cell division in environments very different from their tissue of origin. We suggest that mitotic cell rounding, aided by several actin-regulatory oncogenes, may facilitate this process in a robust, context-independent manner.

Two-step interphase microtubule disassembly aids spindle morphogenesis

BackgroundEntry into mitosis triggers profound changes in cell shape and cytoskeletal organisation. Here, by studying microtubule remodelling in human flat mitotic cells, we identify a two-step process of interphase microtubule disassembly.ResultsFirst, a microtubule-stabilising protein, Ensconsin/MAP7, is inactivated in prophase as a consequence of its phosphorylation downstream of Cdk1/cyclin B. This leads to a reduction in interphase microtubule stability that may help to fuel the growth of centrosomally nucleated microtubules. The peripheral interphase microtubules that remain are then rapidly lost as the concentration of tubulin heterodimers falls following dissolution of the nuclear compartment boundary. Finally, we show that a failure to destabilise microtubules in prophase leads to the formation of microtubule clumps, which interfere with spindle assembly.ConclusionsThis analysis highlights the importance of the step-wise remodelling of the microtubule cytoskeleton and the significance of permeabilisation of the nuclear envelope in coordinating the changes in cellular organisation and biochemistry that accompany mitotic entry.

Role of Wnt signaling in neural crest development: from induction to migration

Several inductive signals are required for NC induction, such as BMPs, Wnts, FGFs and RA; however we do not have a clear picture of how the NC is induced in a precise location along the medio-lateral and anterior-posterior axis of the embryo. By performing graft experiments as well as gain and loss of function experiments of different signals, we have shown that dorso-lateral mesoderm induces NC by producing Wnt8; while prechordal mesoderm inhibits NC at the anterior neural fold by secreting Dkk1.