Biology subjects

Hoogenraad, C. C.

Publications and source records attributed to Hoogenraad, C. C..

3 recordsLinked to original sources

bioRxiv2018

MAP7 family proteins are microtubule-tethered allosteric activators of kinesin-1.

Kinesin-1 is responsible for microtubule-based transport of numerous cellular cargoes. Here, we explored the regulation of kinesin-1 by MAP7 proteins. We found that all four mammalian MAP7 family members bind to kinesin-1. In HeLa cells, MAP7, MAP7D1 and MAP7D3 act redundantly to enable kinesin-1-dependent transport and microtubule recruitment of the truncated kinesin-1 KIF5B-560, which contains the stalk but not the cargo-binding and autoregulatory regions. In vitro, purified MAP7 and MAP7D3 increase microtubule landing rate and processivity of kinesin-1 through transient association with the motor. MAP7 proteins promote binding of kinesin-1 to microtubules both directly, through the N-terminal microtubule-binding domain and unstructured linker region, and indirectly, through an allosteric effect exerted by the kinesin-binding C-terminal domain. Compared to MAP7, MAP7D3 has a higher affinity for kinesin-1 and a lower affinity for microtubules and, unlike MAP7, can be co-transported with the motor. We propose that MAP7 proteins are microtubule-tethered kinesin-1 activators, with which the motor transiently interacts as it moves along microtubules.\n\nSummaryA combination of experiments in cells and in vitro reconstitution assays demonstrated that mammalian MAP7 family proteins act redundantly to activate kinesin-1 and promote its microtubule binding and processivity by transiently associating with the stalk region of the motor.

cell biology↗

bioRxiv2017

Molecular pathway underlying bouton stabilization by Semaphorin4D during inhibitory synapse formation

Changes in inhibitory connections are essential for experience-dependent circuit adaptations. Defects in inhibitory synapses are linked to neurodevelopmental disorders, but the molecular processes underlying inhibitory synapse formation are not well understood. Here we use high resolution two-photon microscopy in organotypic hippocampal slices to examine the signaling pathways induced by the postsynaptic signaling molecule Semaphorin4D (Sema4D) during inhibitory synapse formation. By monitoring changes in individual GFP-labeled presynaptic boutons we found that the primary action of Sema4D is to induce stabilization of presynaptic boutons within tens of minutes. Stabilizing boutons rapidly recruited synaptic vesicles, which was followed by accumulation of postsynaptic gephyrin. Newly formed inhibitory synapses were complete and functional after 24 hours, as determined by electrophysiology and immunohistochemistry. We further showed that Sema4D signaling is regulated by network activity and can induce a local increase in bouton density, suggesting a possible role in circuit adaptation. We further examined the intracellular signaling cascade triggered by Sema4D and found that bouton stabilization occurred through rapid remodeling of actin, and this could be mimicked by the actin-depolymerizing drug Latrunculin B or by reducing ROCK activity. The intracellular signaling cascade required activation of the receptor tyrosine kinase MET, which is a well-known autism risk factor. Our immunohistochemistry data suggests that MET may be localized to presynaptic inhibitory axons. Together, our data yield important insights in the molecular pathway underlying activity-dependent Sema4D-induced synapse formation and reveal a novel role for MET in inhibitory synapses.\n\nSignificance StatementGABAergic synapses provide the main inhibitory control of neuronal activity in the brain. We make important steps in unraveling the molecular processes that take place when formation of inhibitory synapses is triggered by a specific signaling molecule, Sema4D. We find that this process depends on network activity and involves specific remodeling of the intracellular actin cytoskeleton. We also reveal a previously unknown role for MET in inhibitory synapses. As defects in GABAergic synapses have been implied in many brain disorders, and mutations in MET are strong risk factors for autism, our findings urge for a further investigation of the role of MET at inhibitory synapses.

neuroscience↗

bioRxiv2016

Probing cytoskeletal modulation of passive and active intracellular dynamics using nanobody-functionalized quantum dots

The cytoplasm is a highly complex and heterogeneous medium that is structured by the cytoskeleton. Cytoskeletal organization and dynamics are known to modulate cytoplasmic transport processes, but how local transport dynamics depends on the highly heterogeneous intracellular organization of F-actin and microtubules is poorly understood. Here we use a novel delivery and functionalization strategy to utilize quantum dots (QDs) as probes for transport dynamics in different sub-cellular environments. Rapid imaging of non-functionalized QDs revealed two populations with a 100-fold difference in diffusion constant. Depolymerization of actin increased the fast diffusing fraction, suggesting that slow QDs are trapped inside the actin network. When nanobody-functionalized QDs were targeted to different kinesin motor proteins and moved over microtubules, they did not experience strong actin-induced transverse displacements, as suggested previously. Only kinesin-1 bound QDs displayed subtle directional fluctuations, because the specific subset of stable microtubules used by this motor underwent prominent undulations. Using actin-targeting agents we found that the actin network suppresses most microtubule shape remodeling, rather than promoting them. These results demonstrate how the spatial and structural heterogeneity of the cytoskeleton imposes large variations in non-equilibrium intracellular dynamics.

biophysics↗