Schizosaccharomyces pombe kinesin-5 switches direction using a steric blocking mechanism.

Cut7, the sole kinesin-5 in Schizosaccharomyces pombe, is essential for mitosis. Like other yeast kinesin-5 motors, Cut7 can reverse its stepping direction, by mechanisms that are currently unclear. Here we show that for full-length Cut7, the key determinant of stepping direction is the degree of motor crowding on the microtubule lattice, with greater crowding converting the motor from minus end-directed to plus end-directed stepping. To explain how high Cut7 occupancy causes this reversal, we postulate a simple proximity sensing mechanism that operates via steric blocking. We propose that the minus end-directed stepping action of Cut7 is selectively inhibited by collisions with neighbors under crowded conditions, whereas its plus end-directed action, being less space-hungry, is not. In support of this idea, we show that the direction of Cut7-driven microtubule sliding can be reversed by crowding it with non-Cut7 proteins. Thus, crowding by either dynein microtubule binding domain or Klp2, a kinesin-14, converts Cut7 from net minus end-directed to net plus end-directed stepping. Biochemical assays confirm that the Cut7 N terminus increases Cut7 occupancy by binding directly to microtubules. Direct observation by cryoEM reveals that this occupancy-enhancing N-terminal domain is partially ordered. Overall, our data point to a steric blocking mechanism for directional reversal through which collisions of Cut7 motor domains with their neighbors inhibit their minus end-directed stepping action, but not their plus end-directed stepping action. Our model can potentially reconcile a number of previous, apparently conflicting, observations and proposals for the reversal mechanism of yeast kinesins-5.

Data on the regulation of moesin and merlin by the urokinase receptor (uPAR): Model explaining distal activation of integrins by uPAR.

The data presented herein are connected to our research article (doi: 10.1016/j.biocel.2017.04.012) [1], in which we investigated the functional connections between the urokinase receptor (uPAR), and the ezrin/radixin/moesin (ERM) proteins, moesin and merlin [1]. Firstly, a model of action is proposed that enlightens how uPAR regulates distal integrins. In addition, data show the effects of expressing wild-type moesin or permanently active T558D mutant of moesin on angiogenesis and morphology of human aortic endothelial cells (HAEC). Additional data compare the effects of urokinase (uPA, the main ligand of uPAR) on the same cells. Lastly, we provide technical data demonstrating the effects of specific siRNA for moesin and merlin on moesin and merlin expression, respectively.

Moesin and merlin regulate urokinase receptor-dependent endothelial cell migration, adhesion and angiogenesis.

The glycosyl-phosphatidyl-inositol (GPI)-anchored urokinase receptor (uPAR) has no intracellular domain, but nevertheless initiates signalling through proximal interactions with other membrane receptors including integrins. The relationships between uPAR and ezrin/radixin/moesin (ERM) proteins, moesin and merlin have never been explored. Moesin and merlin are versatile membrane-actin links and regulators of receptors signalling, respectively. We show that uPAR controls moesin and merlin, which propagate uPAR-initiated signals and modulate integrin functions, thereby regulating uPAR activity. uPAR rapidly de-phosphorylates moesin and phosphorylates merlin inactivating both proteins, and enhancing cell migration and angiogenesis. Moesin behaves as a molecular switch turning either on or off uPAR signalling through cycles of de-activation/activation, or sustained activation, respectively. Furthermore, moesin is at the crossroads of uPAR-initiated outside-in and inside-out signalling promoting integrin-dependent cell adhesion suggesting that uPAR also activates integrins distally through moesin. Knocking down merlin expression enhanced cell migration and adhesion through different regulation of fibronectin- and vitronectin-binding integrins.

The urokinase receptor in the central nervous system.

The urokinase receptor (uPAR) is a multifunctional glycosylphosphatidylinositol-anchored protein that regulates important processes such as gene expression, cell proliferation, adhesion, migration, invasion, and metastasis. uPAR is an essential component of the plasminogen activation cascade, a protease receptor that binds the urokinase-type plasminogen activator. uPAR is also an adhesion-modulating receptor, and a signalling receptor transmitting signals to the cell through lateral interactions with a wide array of membrane receptors. Altogether, the external ligands and membrane-bound partners of uPAR constitute a rich uPAR interactome. Recently, a new ligand of uPAR has been identified as the SRPX2 protein which is essential in language and cognitive development. SRPX2 is the second identified gene involved in language disorders. However, previous studies revealed cognitive disorders and defects in the development of the GABAergic interneurons in uPAR null mice. In addition, the expression of uPAR correlates with important human diseases such as epilepsy, autism, multiple sclerosis, Alzheimer's, AIDS dementia, cerebral malaria, and brain tumours. Therefore, uPAR has unexpectedly become a significant receptor in the central nervous system and made a few steps into philosophy. Language is indeed intimately linked to human culture. This in-depth review presents the structure and the sequences of uPAR that are essential for drug design and the generation of new inhibitors. In addition, we summarize all the inhibitors of uPAR that have been created so far. Finally, we discuss the functions of uPAR in the development, functioning, and pathology of the central nervous system.