On the lipid mediated regulation of the cell adhesion protein vinculin

Cellular events such as embryogenesis and tissue formation require directed cell adhesion and migration. Focal adhesions are the major cell-matrix connections that link the extracellular matrix glycoproteins to the actin cytoskeleton via the transmembrane receptors integrins. The cytoplasmic domain of integrins interacts with several cytoskeletal and regulatory proteins that are involved in the assembly of the adhesion complex and help in the functional regulation of the adhesion dynamics. In these adhesion complexes, vinculin acts as a molecular scaffold since it interacts with numerous ligands and serves as a connecting link between the membrane and the actin cytoskeleton. Acidic phospholipids like PIP2 are known to structurally and functionally modulate vinculin. This work aimed at the analysis of acidic phospholipid mediated regulation of vinculin at the adhesion sites. Design and construction of a lipid binding deficient mutant vinculin-LD allowed to characterize the mode of regulation of vinculin at the focal adhesions. Biochemical analyses showed that vinculin-LD failed to co-sediment with lipid vesicles while leaving its interaction with important ligands like actin and the vinculin head (Vh) intact. Cell biological analyses of vinculin-LD proved that the lipid binding is not required for the focal adhesion targeting as previously suggested. Analysis of the incorporation rates of this mutant by fluorescent recovery after photobleaching (FRAP) also indicated that the vinculin recruitment to focal adhesions is normal. However, cells expressing vinculin-LD had a serious delay in cell adhesion and spreading and the motility was grossly impaired on various extracellular matrices. Thus, PIP2 interaction with vinculin is not essential for its adhesion localization but vinculin acts as a lipid sensor greatly influencing the adhesion site dynamics mediated through PIP2.Für die Anheftung von Zellen an die zugrundeliegende Matrix sowie für die Migration, bspw. während der Embryogenese oder bei der Gewebebildung, ist die Ausbildung von sog. Fokalkontakten von großer Bedeutung. Dabei werden die Glycoproteine der extrazellulären Matrix über den Transmembranrezeptor Integrin mit dem Aktin Zytoskelett verbunden. Die zytoplasmatische Domäne des Integrins interagiert mit verschiedenen Zytoskelett- und Regulatorproteinen, die an der Assemblierung des Adhäsionskomplexes und/oder der funktionalen Regulation der Adhäsionsdynamik beteiligt sind. In diesen Adhäsionskomplexen übernimmt Vinculin die Rolle eines molekularen Gerüsts, das mit vielen Liganden interagiert und die Membran mit dem Aktin Zytoskelett verbindet. Saure Phospholipide wie PIP2 tragen außerdem zur strukturellen und funktionellen Modulation von Vinculin bei. Diese Arbeit untersucht die Rolle der sauren Phospholipide bei der Regulation von Vinculin an den Fokalkontakten. Eine nähere Charakterisierung erfolgte nach Design und Konstruktion einer Lipidbindungs-defizienten Mutante, Vinculin-LD. Biochemische Analysen zeigen, dass Vinculin-LD nicht mit Lipid Vesikeln kosedimentiert, die Interaktionen mit wichtigen Liganden wie Aktin oder Vinculin Kopf (Vh) jedoch intakt sind. Zellbiologische Untersuchungen mit Vinculin-LD zeigen, dass die Lipidbindung nicht, wie vermutet, maßgeblich für die Rekrutierung in Fokalkontakte ist. Eine Analyse der Einbauraten der Vinculin-LD Mutante mittels Fluorescence-Recovery after Photobleaching (FRAP) deutet auf eine normale Rekrutierung von Vinculin zu den Fokaladhäsionen hin. Zellen, die Vinculin-LD rekrutierten, zeigen jedoch eine Verzögerung bei der Zelladhäsion, der Ausbreitung und in ihrer Beweglichkeit auf diversen extrazellulären Matrices.PIP2 ist nicht essentiell für die Lokalisierung von Vinculin in Fokalkontakte. Vinculin fungiert als Lipid-Sensor, der die PIP2 abhängige Dynamik der Adhäsionskontakte stark beeinflusst

Myosin II regulates activity dependent compensatory endocytosis at central synapses

Recent evidence suggests that endocytosis, not exocytosis, can be rate limiting for neurotransmitter release at excitatory CNS synapses during sustained activity and therefore may be a principal determinant of synaptic fatigue. At low stimulation frequencies, the probability of synaptic release is linked to the probability of synaptic retrieval such that evoked release results in proportional retrieval even for release of single synaptic vesicles. The exact mechanism by which the retrieval rates are coupled to release rates, known as compensatory endocytosis, remains unknown. Here we show that inactivation of presynaptic myosin II (MII) decreases the probability of synaptic retrieval. To be able to differentiate between the presynaptic and postsynaptic functions of MII, we developed a live cell substrate patterning technique to create defined neural circuits composed of small numbers of embryonic mouse hippocampal neurons and physically isolated from the surrounding culture. Acute application of blebbistatin to inactivate MII in circuits strongly inhibited evoked release but not spontaneous release. In circuits incorporating both control and MIIB knock-out cells, loss of presynaptic MIIB function correlated with a large decrease in the amplitude of evoked release. Using activity-dependent markers FM1–43 and horseradish peroxidase, we found that MII inactivation greatly slowed vesicular replenishment of the recycling pool but did not impede synaptic release. These results indicate that MII-driven tension or actin dynamics regulate the major pathway for synaptic vesicle retrieval. Changes in retrieval rates determine the size of the recycling pool. The resulting effect on release rates, in turn, brings about changes in synaptic strength

Conditional Myh9 and Myh10 inactivation in adult mouse renal epithelium results in progressive kidney disease

Actin-associated nonmuscle myosin II (NM2) motor proteins play critical roles in a myriad of cellular functions, including endocytosis and organelle transport pathways. Cell type–specific expression and unique subcellular localization of the NM2 proteins, encoded by the Myh9 and Myh10 genes, in the mouse kidney tubules led us to hypothesize that these proteins have specialized functional roles within the renal epithelium. Inducible conditional knockout (cKO) of Myh9 and Myh10 in the renal tubules of adult mice resulted in progressive kidney disease. Prior to overt renal tubular injury, we observed intracellular accumulation of the glycosylphosphatidylinositol-anchored protein uromodulin (UMOD) and gradual loss of Na+ K+ 2Cl– cotransporter from the apical membrane of the thick ascending limb epithelia. The UMOD accumulation coincided with expansion of endoplasmic reticulum (ER) tubules and activation of ER stress and unfolded protein response pathways in Myh9&10-cKO kidneys. We conclude that NM2 proteins are required for localization and transport of UMOD and loss of function results in accumulation of UMOD and ER stress–mediated progressive renal tubulointerstitial disease. These observations establish cell type–specific role(s) for NM2 proteins in regulation of specialized renal epithelial transport pathways and reveal the possibility that human kidney disease associated with MYH9 mutations could be of renal epithelial origin

Nerve growth factor stimulates axon outgrowth through negative regulation of growth cone actomyosin restraint of microtubule advance

Nerve growth factor (NGF) promotes growth, differentiation, and survival of sensory neurons in the mammalian nervous system. Little is known about how NGF elicits faster axon outgrowth or how growth cones integrate and transform signal input to motor output. Using cultured mouse dorsal root ganglion neurons, we found that myosin II (MII) is required for NGF to stimulate faster axon outgrowth. From experiments inducing loss or gain of function of MII, specific MII isoforms, and vinculin-dependent adhesion-cytoskeletal coupling, we determined that NGF causes decreased vinculin-dependent actomyosin restraint of microtubule advance. Inhibition of MII blocked NGF stimulation, indicating the central role of restraint in directed outgrowth. The restraint consists of myosin IIB- and IIA-dependent processes: retrograde actin network flow and transverse actin bundling, respectively. The processes differentially contribute on laminin-1 and fibronectin due to selective actin tethering to adhesions. On laminin-1, NGF induced greater vinculin-dependent adhesion–cytoskeletal coupling, which slowed retrograde actin network flow (i.e., it regulated the molecular clutch). On fibronectin, NGF caused inactivation of myosin IIA, which negatively regulated actin bundling. On both substrates, the result was the same: NGF-induced weakening of MII-dependent restraint led to dynamic microtubules entering the actin-rich periphery more frequently, giving rise to faster elongation

Loss-of-Function Mutations in FRRS1L Lead to an Epileptic-Dyskinetic Encephalopathy

Glutamatergic neurotransmission governs excitatory signaling in the mammalian brain, and abnormalities of glutamate signaling have been shown to contribute to both epilepsy and hyperkinetic movement disorders. The etiology of many severe childhood movement disorders and epilepsies remains uncharacterized. We describe a neurological disorder with epilepsy and prominent choreoathetosis caused by biallelic pathogenic variants in FRRS1L, which encodes an AMPA receptor outer-core protein. Loss of FRRS1L function attenuates AMPA-mediated currents, implicating chronic abnormalities of glutamatergic neurotransmission in this monogenic neurological disease of childhood