Functional Inhibition of Katanin Affects Synaptic Plasticity.

Dynamic microtubules critically regulate synaptic functions, but the role of microtubule severing in these processes is barely understood. Katanin is a neuronally expressed microtubule-severing complex regulating microtubule number and length in cell division or neurogenesis; however, its potential role in synaptic functions has remained unknown. Studying mice from both sexes, we found that katanin is abundant in neuronal dendrites and can be detected at individual excitatory spine synapses. Overexpression of a dominant-negative ATPase-deficient katanin subunit to functionally inhibit severing alters the growth of microtubules in dendrites, specifically at premature but not mature neuronal stages without affecting spine density. Notably, interference with katanin function prevented structural spine remodeling following single synapse glutamate uncaging and significantly affected the potentiation of AMPA-receptor-mediated excitatory currents after chemical induction of long-term potentiation. Furthermore, katanin inhibition reduced the invasion of microtubules into fully developed spines. Our data demonstrate that katanin-mediated microtubule severing regulates structural and functional plasticity at synaptic sites.

The kinesin Kif21b binds myosin Va and mediates changes in actin dynamics underlying homeostatic synaptic downscaling.

Homeostatic synaptic plasticity adjusts the strength of synapses to restrain neuronal activity within a physiological range. Postsynaptic guanylate kinase-associated protein (GKAP) controls the bidirectional synaptic scaling of AMPA receptors (AMPARs); however, mechanisms by which chronic activity triggers cytoskeletal remodeling to downscale synaptic transmission are barely understood. Here, we report that the microtubule-dependent kinesin motor Kif21b binds GKAP and likewise is located in dendritic spines in a myosin Va- and neuronal-activity-dependent manner. Kif21b depletion unexpectedly alters actin dynamics in spines, and adaptation of actin turnover following chronic activity is lost in Kif21b-knockout neurons. Consistent with a role of the kinesin in regulating actin dynamics, Kif21b overexpression promotes actin polymerization. Moreover, Kif21b controls GKAP removal from spines and the decrease of GluA2-containing AMPARs from the neuronal surface, thereby inducing homeostatic synaptic downscaling. Our data highlight a critical role of Kif21b at the synaptic actin cytoskeleton underlying homeostatic scaling of neuronal firing.

Muskelin regulates actin-dependent synaptic changes and intrinsic brain activity relevant to behavioral and cognitive processes.

Muskelin (Mkln1) is implicated in neuronal function, regulating plasma membrane receptor trafficking. However, its influence on intrinsic brain activity and corresponding behavioral processes remains unclear. Here we show that murine Mkln1 knockout causes non-habituating locomotor activity, increased exploratory drive, and decreased locomotor response to amphetamine. Muskelin deficiency impairs social novelty detection while promoting the retention of spatial reference memory and fear extinction recall. This is strongly mirrored in either weaker or stronger resting-state functional connectivity between critical circuits mediating locomotor exploration and cognition. We show that Mkln1 deletion alters dendrite branching and spine structure, coinciding with enhanced AMPAR-mediated synaptic transmission but selective impairment in synaptic potentiation maintenance. We identify muskelin at excitatory synapses and highlight its role in regulating dendritic spine actin stability. Our findings point to aberrant spine actin modulation and changes in glutamatergic synaptic function as critical mechanisms that contribute to the neurobehavioral phenotype arising from Mkln1 ablation.

Spastin depletion increases tubulin polyglutamylation and impairs kinesin-mediated neuronal transport, leading to working and associative memory deficits.

Mutations in the gene encoding the microtubule-severing protein spastin (spastic paraplegia 4 [SPG4]) cause hereditary spastic paraplegia (HSP), associated with neurodegeneration, spasticity, and motor impairment. Complicated forms (complicated HSP [cHSP]) further include cognitive deficits and dementia; however, the etiology and dysfunctional mechanisms of cHSP have remained unknown. Here, we report specific working and associative memory deficits upon spastin depletion in mice. Loss of spastin-mediated severing leads to reduced synapse numbers, accompanied by lower miniature excitatory postsynaptic current (mEPSC) frequencies. At the subcellular level, mutant neurons are characterized by longer microtubules with increased tubulin polyglutamylation levels. Notably, these conditions reduce kinesin-microtubule binding, impair the processivity of kinesin family protein (KIF) 5, and reduce the delivery of presynaptic vesicles and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Rescue experiments confirm the specificity of these results by showing that wild-type spastin, but not the severing-deficient and disease-associated K388R mutant, normalizes the effects at the synaptic, microtubule, and transport levels. In addition, short hairpin RNA (shRNA)-mediated reduction of tubulin polyglutamylation on spastin knockout background normalizes KIF5 transport deficits and attenuates the loss of excitatory synapses. Our data provide a mechanism that connects spastin dysfunction with the regulation of kinesin-mediated cargo transport, synapse integrity, and cognition.

Myosin VI Drives Clathrin-Mediated AMPA Receptor Endocytosis to Facilitate Cerebellar Long-Term Depression.

Myosin VI is an actin-based cytoskeletal motor implicated in various steps of membrane trafficking. Here, we investigated whether this myosin is crucial for synaptic function and plasticity in neurons. We find that myosin VI localizes at cerebellar parallel fiber to Purkinje cell synapses and that the myosin is indispensable for long-term depression of AMPA-receptor-mediated synaptic signal transmission at this synapse. Moreover, direct visualization of GluA2-containing AMPA receptors in Purkinje cells reveals that the myosin drives removal of AMPA receptors from the surface of dendritic spines in an activity-dependent manner. Co-immunoprecipitation and super-resolution microscopy indicate that specifically the interaction of myosin VI with the clathrin adaptor component α-adaptin is important during long-term depression. Together, these data suggest that myosin VI directly promotes clathrin-mediated endocytosis of AMPA receptors in Purkinje cells to mediate cerebellar long-term depression. Our results provide insights into myosin VI function and the molecular mechanisms underlying synaptic plasticity.

Neurobeachin and the Kinesin KIF21B Are Critical for Endocytic Recycling of NMDA Receptors and Regulate Social Behavior.

Autism spectrum disorders (ASDs) are associated with mutations affecting synaptic components, including GluN2B-NMDA receptors (NMDARs) and neurobeachin (NBEA). NBEA participates in biosynthetic pathways to regulate synapse receptor targeting, synaptic function, cognition, and social behavior. However, the role of NBEA-mediated transport in specific trafficking routes is unclear. Here, we highlight an additional function for NBEA in the local delivery and surface re-insertion of synaptic receptors in mouse neurons. NBEA dynamically interacts with Rab4-positive recycling endosomes, transiently enters spines in an activity-dependent manner, and regulates GluN2B-NMDAR recycling. Furthermore, we show that the microtubule growth inhibitor kinesin KIF21B constrains NBEA dynamics and is present in the NBEA-recycling endosome-NMDAR complex. Notably, Kif21b knockout decreases NMDAR surface expression and alters social behavior in mice, consistent with reported social deficits in Nbea mutants. The influence of NBEA-KIF21B interactions on GluN2B-NMDAR local recycling may be relevant to mechanisms underlying ASD etiology.

The Kinesin KIF21B Regulates Microtubule Dynamics and Is Essential for Neuronal Morphology, Synapse Function, and Learning and Memory.

The kinesin KIF21B is implicated in several human neurological disorders, including delayed cognitive development, yet it remains unclear how KIF21B dysfunction may contribute to pathology. One limitation is that relatively little is known about KIF21B-mediated physiological functions. Here, we generated Kif21b knockout mice and used cellular assays to investigate the relevance of KIF21B in neuronal and in vivo function. We show that KIF21B is a processive motor protein and identify an additional role for KIF21B in regulating microtubule dynamics. In neurons lacking KIF21B, microtubules grow more slowly and persistently, leading to tighter packing in dendrites. KIF21B-deficient neurons exhibit decreased dendritic arbor complexity and reduced spine density, which correlate with deficits in synaptic transmission. Consistent with these observations, Kif21b-null mice exhibit behavioral changes involving learning and memory deficits. Our study provides insight into the cellular function of KIF21B and the basis for cognitive decline resulting from KIF21B dysregulation.

GSK3 and KIF5 regulate activity-dependent sorting of gephyrin between axons and dendrites.

The kinesin KIF5 transports neuronal cargoes into axons and dendrites. Isolated KIF5 motor domains preferentially move into axons, however KIF5 binding to GRIP1 or gephyrin drives the motor into dendrites, to deliver AMPA receptors (AMPARs) or glycine receptors (GlyRs), respectively. At postsynaptic sites, gephyrin forms a multimeric scaffold to anchor GlyRs and GABAA receptors (GABAARs) in apposition to inhibitory presynaptic terminals. Here, we report the unexpected observation that increased intracellular calcium through chronic activation of AMPARs, steers a newly synthesized gephyrin fusion protein (tomato-gephyrin) to axons and interferes with its normal delivery into dendrites of cultured neurons. Axonal gephyrin clusters were not apposed to presynaptic terminals, but colocalized with GlyRs and neuroligin-2 (NLG2). Notably, functional blockade of glycogen synthase kinase-3 (GSK3) and KIF5 normalized gephyrin missorting into the axonal compartment. In contrast, mutagenesis of gephyrin S270, a GSK3 target, did not contribute to axo-dendritic sorting. Our data are consistent with previous observations, which report regulation of kinesin motility through GSK3 activity. They suggest that GSK3 regulates the sorting of GlyR/gephyrin and NLG2 complexes in a KIF5-dependent manner.

GRIP1 interlinks N-cadherin and AMPA receptors at vesicles to promote combined cargo transport into dendrites.

The GluA2 subunit of AMPA-type glutamate receptors (AMPARs) regulates excitatory synaptic transmission in neurons. In addition, the transsynaptic cell adhesion molecule N-cadherin controls excitatory synapse function and stabilizes dendritic spine structures. At postsynaptic membranes, GluA2 physically binds N-cadherin, underlying spine growth and synaptic modulation. We report that N-cadherin binds to PSD-95/SAP90/DLG/ZO-1 (PDZ) domain 2 of the glutamate receptor interacting protein 1 (GRIP1) through its intracellular C terminus. N-cadherin and GluA2-containing AMPARs are presorted to identical transport vesicles for dendrite delivery, and live imaging reveals cotransport of both proteins. The kinesin KIF5 powers GluA2/N-cadherin codelivery by using GRIP1 as a multilink interface. Notably, GluA2 and N-cadherin use different PDZ domains on GRIP1 to simultaneously bind the transport complex, and interference with either binding motif impairs the turnover of both synaptic cargoes. Depolymerization of microtubules, deletion of the KIF5 motor domain, or specific blockade of AMPAR exocytosis affects delivery of GluA2/N-cadherin vesicles. At the functional level, interference with this cotransport reduces the number of spine protrusions and excitatory synapses. Our data suggest the concept that the multi-PDZ-domain adaptor protein GRIP1 can act as a scaffold at trafficking vesicles in the combined delivery of AMPARs and N-cadherin into dendrites.

Covalent quantum dot receptor linkage via the acyl carrier protein for single-molecule tracking, internalization, and trafficking studies.

Here we describe a labeling technique for the covalent linkage of quantum dots to transmembrane receptors for single-molecule tracking. Our method combines the acyl carrier protein (ACP) technique with coenzyme A (CoA)-functionalized quantum dots to covalently attach quantum dots to ACP fusions of receptor proteins. The advantages of this approach include: (i) the use of a smaller attachment linker than in many other quantum dot-labeling systems; (ii) the ability to achieve a reliable 1:1 fluorophore-to-receptor labeling stoichiometry; (iii) the specificity of the method; and (iv) the covalent nature of the quantum dot linkage. We demonstrate the general suitability of this technique in single-molecule tracking, internalization, and trafficking studies by imaging two different transmembrane receptors in living cells.

Step-wise formation of eukaryotic double-row polyribosomes and circular translation of polysomal mRNA.

The time course of polysome formation was studied in a long-term wheat germ cell-free translation system using sedimentation and electron microscopy techniques. The polysomes were formed on uncapped luciferase mRNA with translation-enhancing 5' and 3' UTRs. The formation of fully loaded polysomes was found to be a long process that required many rounds of translation and proceeded via several phases. First, short linear polysomes containing no more than six ribosomes were formed. Next, folding of these polysomes into short double-row clusters occurred. Subsequent gradual elongation of the clusters gave rise to heavy-loaded double-row strings containing up to 30-40 ribosomes. The formation of the double-row polysomes was considered to be equivalent to circularization of polysomes, with antiparallel halves of the circle being laterally stuck together by ribosome interactions. A slow exchange with free ribosomes and free mRNA observed in the double-row type polysomes, as well as the resistance of translation in them to AMP-PNP, provided evidence that most polysomal ribosomes reinitiate translation within the circularized polysomes without scanning of 5' UTR, while de novo initiation including 5' UTR scanning proceeds at a much slower rate. Removal or replacements of 5' and 3' UTRs affected the initial phase of translation, but did not prevent the formation of the double-row polysomes during translation.

Visualizing Smad1/4 signaling response to bone morphogenetic protein-4 activation by FRET biosensors.

Smad proteins are the major signal transducers for the Transforming Growth Factor superfamily of cytokines and their serine/threonine kinase receptors. Smads mediate the signal from the membrane into the nucleus. Bone Morphogenetic Protein-4 stimulates phosphorylation of Smad1, which interacts with Smad4. This complex translocates into the nucleus and regulates transcription of target genes. Here, we report our development of cellular fluorescence biosensors for direct visualization of Smad signaling in live mammalian cells. Fluorescence resonance energy transfer between cyan and yellow fluorescent proteins fused to the Smad1 and Smad4 proteins was used to unravel the temporal aspects of BMP/Smad signaling. A rate-limiting delay of 2-5 min occurred between BMP activation and Smad1 activity. A similar delay was observed in the Smad1/Smad4 complexation. Further experimentation indicated that the delay is dependent on the MH1 domain and linker of Smad1. These results give new insights into the dynamics of the BMP receptor -Smad1/4 signaling process and provide a new tool for studying Smads.