Hiroki Umeshima

Microtubule-based nuclear movement occurs independently of centrosome positioning in migrating neurons.

During neuronal migration in the developing brain, it is thought that the centrosome precedes the nucleus and provides a cue for nuclear migration along the microtubules. In time-lapse imaging studies of radially migrating granule cells in mouse cerebellar slices, we observed that the movements of the nucleus and centrosome appeared to occur independently of each other. The nucleus often migrated ahead of the centrosome during its saltatory movement, negating the supposed role of the centrosome in pulling the nucleus. The nucleus was associated with dynamic microtubules enveloping the entire nucleus and stable microtubules extending from the leading process to the anterior part of the nucleus. Neither of these perinuclear microtubules converged at the centrosome. Disruption or excess formation of stable microtubules attenuated nuclear migration, indicating that the configuration of stable microtubules is crucial for nuclear migration. The inhibition of LIS1 function, a regulator of a microtubule motor dynein, specifically blocks nuclear migration without affecting the coupling of the centrosome and microtubules in the leading process, suggesting that movements of the nucleus and centrosome are differentially regulated by dynein motor function. Thus, the nucleus moves along the microtubules independently of the position of the centrosome in migrating neurons.

Inhibition of calpain increases LIS1 expression and partially rescues in vivo phenotypes in a mouse model of lissencephaly

Lissencephaly is a devastating neurological disorder caused by defective neuronal migration. LIS1 (official symbol PAFAH1B1, for platelet-activating factor acetylhydrolase, isoform 1b, subunit 1) was identified as the gene mutated in individuals with lissencephaly, and it was found to regulate cytoplasmic dynein function and localization. Here we show that inhibition or knockdown of calpains protects LIS1 from proteolysis, resulting in the augmentation of LIS1 amounts in Lis1+/− mouse embryonic fibroblast cells and rescue of the aberrant distribution of cytoplasmic dynein, mitochondria and β-COP–positive vesicles. We also show that calpain inhibitors improve neuronal migration of Lis1+/− cerebellar granular neurons. Intraperitoneal injection of the calpain inhibitor ALLN to pregnant Lis1+/− dams rescued apoptotic neuronal cell death and neuronal migration defects in Lis1+/− offspring. Furthermore, in utero knockdown of calpain by short hairpin RNA rescued defective cortical layering in Lis1+/− mice. Thus, calpain inhibition is a potential therapeutic intervention for lissencephaly.

Dual phases of migration of cerebellar granule cells guided by axonal and dendritic leading processes

During lamination of the cerebellar cortex, granule cells initially migrate tangentially along the external granule layer, and then make a vertical turn and migrate radially to the internal granule layer. We comparatively analyzed the properties of biphasic migration of granule cells in a microexplant culture in which quantitation of morphology and subcellular localization of molecules were readily accomplished. Tangential migration was guided by a leading process that later formed a parallel fiber axon. Translocation of the soma within the axonal process occurred independently of the rapid displacement of the large growth cone. On the other hand, radial migration was guided by a leading process that differentiated into a dendrite after completion of migration. Displacement of the soma and the tiny growth cone were linked so that the radial leading process adopted locomotion and kept a constant length. We propose that the dual phases of granule cell migration are achieved by distinct cellular mechanisms guided by the leading processes forming an axon and a dendrite, respectively.

Control of Spontaneous Ca2+ Transients Is Critical for Neuronal Maturation in the Developing Neocortex

Neural activity plays roles in the later stages of development of cortical excitatory neurons, including dendritic and axonal arborization, remodeling, and synaptogenesis. However, its role in earlier stages, such as migration and dendritogenesis, is less clear. Here we investigated roles of neural activity in the maturation of cortical neurons, using calcium imaging and expression of prokaryotic voltage-gated sodium channel, NaChBac. Calcium imaging experiments showed that postmigratory neurons in layer II/III exhibited more frequent spontaneous calcium transients than migrating neurons. To test whether such an increase of neural activity may promote neuronal maturation, we elevated the activity of migrating neurons by NaChBac expression. Elevation of neural activity impeded migration, and induced premature branching of the leading process before neurons arrived at layer II/III. Many NaChBac-expressing neurons in deep cortical layers were not attached to radial glial fibers, suggesting that these neurons had stopped migration. Morphological and immunohistochemical analyses suggested that branched leading processes of NaChBac-expressing neurons differentiated into dendrites. Our results suggest that developmental control of spontaneous calcium transients is critical for maturation of cortical excitatory neurons in vivo: keeping cellular excitability low is important for migration, and increasing spontaneous neural activity may stop migration and promote dendrite formation.

Differential roles of cyclin-dependent kinase 5 in tangential and radial migration of cerebellar granule cells.

The cerebellar granule cell is a unique neuron which undergoes tangential migration along axonal tracts and radial migration along glial fibers sequentially during postnatal development. Little is known about molecular bases of the differential kinetics of tangential and radial migration. Here we developed a time-lapse imaging assay for tangential migration of cerebellar granule cells, and investigated comparative contributions of cyclin-dependent kinase 5 (CDK5), a key regulator of neuronal migration, in tangential and radial migration of granule cells in vivo and in organotypic cultures. Overexpression of a dominant-negative form of CDK5 severely disrupted cell morphology and somal movement during radial migration, while it only moderately affected tangential migration. Dominant-negative inhibition of CDK5 induced formation of ectopic radial processes in granule cells in vivo which aberrantly elongated into the white matter in the cerebellum. Live imaging of granule cell migration in cerebellar slices revealed that CDK5 regulates not only nuclear migration but also centrosome movement during radial migration. These findings suggest a mode-specific function of CDK5 in neuronal migration.

Caprice/MISP is a novel F-actin bundling protein critical for actin-based cytoskeletal reorganizations

Caprice [C19orf21 actin‐bundling protein in characteristic epithelial cells, also called mitotic interactor and substrate of Plk1 (MISP)] is a novel actin‐related protein identified in the highly‐insoluble subcellular scaffold proteins. This protein contains multiple actin‐binding sites, forms characteristic mesh‐like F‐actin bundles in vitro, and exhibits capricious localization and expression patterns in vivo. Overexpression or knock‐down of Caprice resulted in a dramatic effect on cellular morphology by inducing stress fiber‐like thick filaments or filopodial formations, respectively. Caprice is expressed and localized in distinct cells and tissues with specialized actin‐based structures, such as growth cones of migrating neurons and stereocilia of inner ear hair cells. However, Caprice gene expression is varied among different cell types; especially enriched in several epithelial cells whereas relatively suppressed in a subset of epithelial cells, fibroblasts, and neuroblastoma cells at the transcriptional level. Thus, this protein is expected to be an effector for cell type‐specific actin reorganization with its direct actin‐binding properties and provides a novel model of cell morphology regulation by a non‐ubiquitous single actin‐bundling protein.

Cerebellar granule cells are predominantly generated by terminal symmetric divisions of granule cell precursors

Background: Neurons in the central nervous system (CNS) are generated by symmetric and asymmetric cell division of neural stem cells and their derivative progenitor cells. Cerebellar granule cells are the most abundant neurons in the CNS, and are generated by intensive cell division of granule cell precursors (GCPs) during postnatal development. Dysregulation of GCP cell cycle is causal for some subtypes of medulloblastoma. However, the details and mechanisms underlying neurogenesis from GCPs are not well understood. Results: Using long‐term live‐cell imaging of proliferating GCPs transfected with a fluorescent newborn‐granule cell marker, we found that GCPs underwent predominantly symmetric divisions, generating two GCPs or two neurons, while asymmetric divisions generating a GCP and a neuron were only occasionally observed, in both dissociated culture and within tissues of isolated cerebellar lobules. We found no significant difference in cell cycle length between proliferative and neurogenic divisions, or any consistent changes in cell cycle length during repeated proliferative division. Conclusions: Unlike neural stem cells in the cerebral cortex and spinal cord, which generate many neurons by repeated asymmetric division, cerebellar GCPs produce neurons predominantly by terminal symmetric division. These results indicate diverse mechanisms of neurogenesis in the mammalian brain. Developmental Dynamics 244:748–758, 2015.

Nesprins and opposing microtubule motors generate a point force that drives directional nuclear motion in migrating neurons

ABSTRACT Nuclear migration of newly born neurons is essential for cortex formation in the brain. The nucleus is translocated by actin and microtubules, yet the actual force generated by the interplay of these cytoskeletons remains elusive. High-resolution time-lapse observation of migrating murine cerebellar granule cells revealed that the nucleus actively rotates along the direction of its translocation, independently of centrosome motion. Pharmacological and molecular perturbation indicated that spin torque is primarily generated by microtubule motors through the LINC complex in the absence of actomyosin contractility. In contrast to the prevailing view that microtubules are uniformly oriented around the nucleus, we observed that the perinuclear microtubule arrays are of mixed polarity and both cytoplasmic dynein complex and kinesin-1 are required for nuclear rotation. Kinesin-1 can exert a point force on the nuclear envelope via association with nesprins, and loss of kinesin-1 causes failure in neuronal migration in vivo. Thus, microtubules steer the nucleus and drive its rotation and translocation via a dynamic, focal interaction of nesprins with kinesin-1 and dynein, and this is necessary for neuronal migration during brain development. Highlighted Article: During migration of mouse cerebellar neurons, microtubules dynamically bind to nesprins in the nuclear envelope via kinesin-1 and dynein, and induce sharpening, rotation and translocation of the nucleus independently of actin.

Local traction force in the proximal leading process triggers nuclear translocation during neuronal migration

Somal translocation in long bipolar neurons is regulated by actomyosin contractile forces, yet the precise spatiotemporal sites of force generation are unknown. Here we investigate the force dynamics generated during somal translocation using traction force microscopy. Neurons with a short leading process generated a traction force in the growth cone and counteracting forces in the leading and trailing processes. In contrast, neurons with a long leading process generated a force dipole with opposing traction forces in the proximal leading process during nuclear translocation. Transient accumulation of actin filaments was observed at the dipole center of the two opposing forces, which was abolished by inhibition of myosin II activity. A swelling in the leading process emerged and generated a traction force that pulled the nucleus when nuclear translocation was physically hampered. The traction force in the leading process swelling was uncoupled from somal translocation in neurons expressing a dominant negative mutant of the KASH protein, which disrupts the interaction between cytoskeletal components and the nuclear envelope. Our results suggest that the leading process is the site of generation of actomyosin-dependent traction force in long bipolar neurons, and that the traction force is transmitted to the nucleus via KASH proteins.

Local cell stiffness measurement using probes deformation

In this paper, we propose and demonstrate a new method for local cell stiffness measurement using multiple probes system. Thin glass pipettes were employed to measure the stiffness of cells on a general culture dish. The thin micropipettes were prepared and their spring constant was measured using the Atomic Force Microscopy (AFM) cantilever before stiffness measurement. The deformation of the pipette tips and the cell were observed under an optical microscope. The cell stiffness was calculated from the deformations based on the Hookes low. The measurement results show that the myoblast C2C12 cell on a glass substrate has the stiffness around 0.1 N/m. The experimental results indicate that the proposed method can conduct the time-lapse cell stiffness measurement at multiple points simultaneously.