Role of Nesprin-2 and RanBP2 in BICD2-associated brain developmental disorders.

Bicaudal D2 (BICD2) is responsible for recruiting cytoplasmic dynein to diverse forms of subcellular cargo for their intracellular transport. Mutations in the human BICD2 gene have been found to cause an autosomal dominant form of spinal muscular atrophy (SMA-LED2), and brain developmental defects. Whether and how the latter mutations are related to roles we and others have identified for BICD2 in brain development remains little understood. BICD2 interacts with the nucleoporin RanBP2 to recruit dynein to the nuclear envelope (NE) of Radial Glial Progenitor cells (RGPs) to mediate their well-known but mysterious cell-cycle-regulated interkinetic nuclear migration (INM) behavior, and their subsequent differentiation to form cortical neurons. We more recently found that BICD2 also mediates NE dynein recruitment in migrating post-mitotic neurons, though via a different interactor, Nesprin-2. Here, we report that Nesprin-2 and RanBP2 compete for BICD2-binding in vitro. To test the physiological implications of this behavior, we examined the effects of known BICD2 mutations using in vitro biochemical and in vivo electroporation-mediated brain developmental assays. We find a clear relationship between the ability of BICD2 to bind RanBP2 vs. Nesprin-2 in controlling of nuclear migration and neuronal migration behavior. We propose that mutually exclusive RanBP2-BICD2 vs. Nesprin-2-BICD2 interactions at the NE play successive, critical roles in INM behavior in RGPs and in post-mitotic neuronal migration and errors in these processes contribute to specific human brain malformations.

LIS1 and NudE induce a persistent dynein force-producing state.

Cytoplasmic dynein is responsible for many aspects of cellular and subcellular movement. LIS1, NudE, and NudEL are dynein interactors initially implicated in brain developmental disease but now known to be required in cell migration, nuclear, centrosomal, and microtubule transport, mitosis, and growth cone motility. Identification of a specific role for these proteins in cytoplasmic dynein motor regulation has remained elusive. We find that NudE stably recruits LIS1 to the dynein holoenzyme molecule, where LIS1 interacts with the motor domain during the prepowerstroke state of the dynein crossbridge cycle. NudE abrogates dynein force production, whereas LIS1 alone or with NudE induces a persistent-force dynein state that improves ensemble function of multiple dyneins for transport under high-load conditions. These results likely explain the requirement for LIS1 and NudE in the transport of nuclei, centrosomes, chromosomes, and the microtubule cytoskeleton as well as the particular sensitivity of migrating neurons to reduced LIS1 expression.

Glycogen synthase kinase 3 induces multilineage maturation of human pluripotent stem cell-derived lung progenitors in 3D culture.

Although strategies for directed differentiation of human pluripotent stem cells (hPSCs) into lung and airway have been established, terminal maturation of the cells remains a vexing problem. We show here that in collagen I 3D cultures in the absence of glycogen synthase kinase 3 (GSK3) inhibition, hPSC-derived lung progenitors (LPs) undergo multilineage maturation into proximal cells, type I alveolar epithelial cells and morphologically mature type II cells. Enhanced cell cycling, one of the signaling outputs of GSK3 inhibition, plays a role in the maturation-inhibiting effect of GSK3 inhibition. Using this model, we show NOTCH signaling induced a distal cell fate at the expense of a proximal and ciliated cell fate, whereas WNT signaling promoted a proximal club cell fate, thus implicating both signaling pathways in proximodistal specification in human lung development. These findings establish an approach to achieve multilineage maturation of lung and airway cells from hPSCs, demonstrate a pivotal role of GSK3 in the maturation of lung progenitors and provide novel insight into proximodistal specification during human lung development.

Role of kinesins in directed adenovirus transport and cytoplasmic exploration.

Many viruses, including adenovirus, exhibit bidirectional transport along microtubules following cell entry. Cytoplasmic dynein is responsible for microtubule minus end transport of adenovirus capsids after endosomal escape. However, the identity and roles of the opposing plus end-directed motor(s) remain unknown. We performed an RNAi screen of 38 kinesins, which implicated Kif5B (kinesin-1 family) and additional minor kinesins in adenovirus 5 (Ad5) capsid translocation. Kif5B RNAi markedly increased centrosome accumulation of incoming Ad5 capsids in human A549 pulmonary epithelial cells within the first 30 min post infection, an effect dramatically enhanced by blocking Ad5 nuclear pore targeting using leptomycin B. The Kif5B RNAi phenotype was rescued by expression of RNAi-resistant Kif5A, B, or C, and Kif4A. Kif5B RNAi also inhibited a novel form of microtubule-based "assisted-diffusion" behavior which was apparent between 30 and 60 min p.i. We found the major capsid protein penton base (PB) to recruit kinesin-1, distinct from the hexon role we previously identified for cytoplasmic dynein binding. We propose that adenovirus uses independently recruited kinesin and dynein for directed transport and for a more random microtubule-based assisted diffusion behavior to fully explore the cytoplasm before docking at the nucleus, a mechanism of potential importance for physiological cargoes as well.

Replication of early and recent Zika virus isolates throughout mouse brain development.

Fetal infection with Zika virus (ZIKV) can lead to congenital Zika virus syndrome (cZVS), which includes cortical malformations and microcephaly. The aspects of cortical development that are affected during virus infection are unknown. Using organotypic brain slice cultures generated from embryonic mice of various ages, sites of ZIKV replication including the neocortical proliferative zone and radial columns, as well as the developing midbrain, were identified. The infected radial units are surrounded by uninfected cells undergoing apoptosis, suggesting that programmed cell death may limit viral dissemination in the brain and may constrain virus-associated injury. Therefore, a critical aspect of ZIKV-induced neuropathology may be defined by death of uninfected cells. All ZIKV isolates assayed replicated efficiently in early and midgestation cultures, and two isolates examined replicated in late-gestation tissue. Alteration of neocortical cytoarchitecture, such as disruption of the highly elongated basal processes of the radial glial progenitor cells and impairment of postmitotic neuronal migration, were also observed. These data suggest that all lineages of ZIKV tested are neurotropic, and that ZIKV infection interferes with multiple aspects of neurodevelopment that contribute to the complexity of cZVS.

Conformational changes in the adenovirus hexon subunit responsible for regulating cytoplasmic dynein recruitment.

UNLABELLED: Virus capsids provide genome protection from environmental challenges but are also poised to execute a program of compositional and conformational changes to facilitate virion entry and infection. The most abundant adenovirus serotype 5 (AdV5) capsid protein, hexon, directly recruits the motor protein cytoplasmic dynein following virion entry. Dynein recruitment is crucial for capsid transport to the nucleus and requires the transient exposure of AdV5 hexon to low pH, presumably mimicking passage through the endosomal compartment. These results suggest a pH-dependent capsid modification during early infection. The changes to hexon structure controlling this behavior have not been explored. We report that hexon remains trimeric at low pH but undergoes more subtle conformational changes. These changes are indicated by increased sensitivities to SDS-mediated dissociation and dispase proteolysis. Both effects are reversed at neutral pH, as is dynein binding by low-pH-treated hexon. Dispase cleavage, which we find maps to a specific site within hypervariable region 1 (HVR1) of AdV5 hexon, has no apparent effect on virion entry but completely inhibits its transport to the nucleus. In addition, an AdV5 mutant containing HVR1 of AdV48 is unable to bind dynein and is strongly inhibited in the postentry transport step. These results reveal that conformational changes involving hexon HVR1 are the basis for a novel viral mechanism controlling capsid transport to the nucleus. IMPORTANCE: The adenovirus serotype 5 (AdV5) capsid protein hexon recruits the molecular motor protein cytoplasmic dynein in a pH-dependent manner, a function critical for efficient transport toward the nucleus and AdV5 infectivity. In this work, we describe how low-pH exposure induces reversible structural changes in AdV5 hexon and how these changes affect dynein binding. In addition, we identified a pH-sensitive dispase cleavage site in hexon HVR1, which depends on the same structural changes and furthermore regulates dynein recruitment and capsid redistribution in infected cells. These data provide the first evidence relating long-known but subtle pH-dependent structural changes in hexon to a more recently established essential but poorly understood role in virus transport. These results have broad implications for understanding virus infectivity in general, and our ability to block the recruitment mechanism has potential therapeutic implications as well.

Asymmetric centrosome inheritance maintains neural progenitors in the neocortex.

Asymmetric divisions of radial glia progenitors produce self-renewing radial glia and differentiating cells simultaneously in the ventricular zone (VZ) of the developing neocortex. Whereas differentiating cells leave the VZ to constitute the future neocortex, renewing radial glia progenitors stay in the VZ for subsequent divisions. The differential behaviour of progenitors and their differentiating progeny is essential for neocortical development; however, the mechanisms that ensure these behavioural differences are unclear. Here we show that asymmetric centrosome inheritance regulates the differential behaviour of renewing progenitors and their differentiating progeny in the embryonic mouse neocortex. Centrosome duplication in dividing radial glia progenitors generates a pair of centrosomes with differently aged mother centrioles. During peak phases of neurogenesis, the centrosome retaining the old mother centriole stays in the VZ and is preferentially inherited by radial glia progenitors, whereas the centrosome containing the new mother centriole mostly leaves the VZ and is largely associated with differentiating cells. Removal of ninein, a mature centriole-specific protein, disrupts the asymmetric segregation and inheritance of the centrosome and causes premature depletion of progenitors from the VZ. These results indicate that preferential inheritance of the centrosome with the mature older mother centriole is required for maintaining radial glia progenitors in the developing mammalian neocortex.

Molecular mechanism for recognition of the cargo adapter Rab6GTP by the dynein adapter BicD2.

Rab6 is a key modulator of protein secretion. The dynein adapter Bicaudal D2 (BicD2) recruits the motors cytoplasmic dynein and kinesin-1 to Rab6GTP-positive vesicles for transport; however, it is unknown how BicD2 recognizes Rab6. Here, we establish a structural model for recognition of Rab6GTP by BicD2, using structure prediction and mutagenesis. The binding site of BicD2 spans two regions of Rab6 that undergo structural changes upon the transition from the GDP- to GTP-bound state, and several hydrophobic interface residues are rearranged, explaining the increased affinity of the active GTP-bound state. Mutations of Rab6GTP that abolish binding to BicD2 also result in reduced co-migration of Rab6GTP/BicD2 in cells, validating our model. These mutations also severely diminished the motility of Rab6-positive vesicles in cells, highlighting the importance of the Rab6GTP/BicD2 interaction for overall motility of the multi-motor complex that contains both kinesin-1 and dynein. Our results provide insights into trafficking of secretory and Golgi-derived vesicles and will help devise therapies for diseases caused by BicD2 mutations, which selectively affect the affinity to Rab6 and other cargoes.

Development and application of in vivo molecular traps reveals that dynein light chain occupancy differentially affects dynein-mediated processes.

The ability to rapidly and specifically regulate protein activity combined with in vivo functional assays and/or imaging can provide unique insight into underlying molecular processes. Here we describe the application of chemically induced dimerization of FKBP to create nearly instantaneous high-affinity bivalent ligands capable of sequestering cellular targets from their endogenous partners. We demonstrate the specificity and efficacy of these inducible, dimeric "traps" for the dynein light chains LC8 (Dynll1) and TcTex1 (Dynlt1). Both light chains can simultaneously bind at adjacent sites of dynein intermediate chain at the base of the dynein motor complex, yet their specific function with respect to the dynein motor or other interacting proteins has been difficult to dissect. Using these traps in cultured mammalian cells, we observed that induction of dimerization of either the LC8 or TcTex1 trap rapidly disrupted early endosomal and lysosomal organization. Dimerization of either trap also disrupted Golgi organization, but at a substantially slower rate. Using either trap, the time course for disruption of each organelle was similar, suggesting a common regulatory mechanism. However, despite the essential role of dynein in cell division, neither trap had a discernable effect on mitotic progression. Taken together, these studies suggest that LC occupancy of the dynein motor complex directly affects some, but not all, dynein-mediated processes. Although the described traps offer a method for rapid inhibition of dynein function, the design principle can be extended to other molecular complexes for in vivo studies.

A two-kinesin mechanism controls neurogenesis in the developing brain.

During the course of brain development, Radial Glial Progenitor (RGP) cells give rise to most of the neurons required for a functional cortex. RGPs can undergo symmetric divisions, which result in RGP duplication, or asymmetric divisions, which result in one RGP as well as one to four neurons. The control of this balance is not fully understood, but must be closely regulated to produce the cells required for a functioning cortex, and to maintain the stem cell pool. In this study, we show that the balance between symmetric and asymmetric RGP divisions is in part regulated by the actions of two kinesins, Kif1A and Kif13B, which we find have opposing roles in neurogenesis through their action on the mitotic spindle in dividing RGPs. We find that Kif1A promotes neurogenesis, whereas Kif13B promotes symmetric, non-neurogenic divisions. Interestingly, the two kinesins are closely related in structure, and members of the same kinesin-3 subfamily, thus their opposing effects on spindle orientation appear to represent a novel mechanism for the regulation of neurogenesis.

NudC is required for interkinetic nuclear migration and neuronal migration during neocortical development.

NudC is a highly conserved protein necessary for cytoplasmic dynein-mediated nuclear migration in Aspergillus nidulans. NudC interacts genetically with Aspergillus NudF and physically with its mammalian orthologue Lis1, which is crucial for nuclear and neuronal migration during brain development. To test for related roles for NudC, we performed in utero electroporation into embryonic rat brain of cDNAs encoding shRNAs as well as wild-type and mutant forms of NudC. We show here that NudC, like Lis1, is required for neuronal migration during neocorticogenesis and we identify a specific role in apical nuclear migration in radial glial progenitor cells. These results identify a novel neuronal migration gene with a specific role in interkinetic nuclear migration, consistent with cytoplasmic dynein regulation.

Mutually exclusive cytoplasmic dynein regulation by NudE-Lis1 and dynactin.

Cytoplasmic dynein is responsible for a wide range of cellular roles. How this single motor protein performs so many functions has remained a major outstanding question for many years. Part of the answer is thought to lie in the diversity of dynein regulators, but how the effects of these factors are coordinated in vivo remains unexplored. We previously found NudE to bind dynein through its light chain 8 (LC8) and intermediate chain (IC) subunits (1), the latter of which also mediates the dynein-dynactin interaction (2). We report here that NudE and dynactin bind to a common region within the IC, and compete for this site. We find LC8 to bind to a novel sequence within NudE, without detectably affecting the dynein-NudE interaction. We further find that commonly used dynein inhibitory reagents have broad effects on the interaction of dynein with its regulatory factors. Together these results reveal an unanticipated mechanism for preventing dual regulation of individual dynein molecules, and identify the IC as a nexus for regulatory interactions within the dynein complex.

NudE and NudEL are required for mitotic progression and are involved in dynein recruitment to kinetochores.

NudE and NudEL are related proteins that interact with cytoplasmic dynein and LIS1. Their functional relationship and involvement in LIS1 and dynein regulation are not completely understood. We find that NudE and NudEL each localize to mitotic kinetochores before dynein, dynactin, ZW10, and LIS1 and exhibit additional temporal and spatial differences in distribution from the motor protein. Inhibition of NudE and NudEL caused metaphase arrest with misoriented chromosomes and defective microtubule attachment. Dynein and dynactin were both displaced from kinetochores by the injection of an anti-NudE/NudEL antibody. Dynein but not dynactin interacted with NudE surprisingly through the dynein intermediate and light chains but not the motor domain. Together, these results identify a common function for NudE and NudEL in mitotic progression and identify an alternative mechanism for dynein recruitment to and regulation at kinetochores.

Dynein at the cortex.

Cytoplasmic dynein is a minus end directed microtubule motor protein with numerous functions during interphase and mitosis. Recent evidence has identified several roles mediated by a fraction of cytoplasmic dynein associated with the cell cortex. So far, these include nuclear migration, mitotic spindle orientation, and cytoskeletal reorientation during wound healing, but others are likely. The possibility that a cortically bound form of dynein might represent its most ancient evolutionary state is discussed.

Role of the kinetochore/cell cycle checkpoint protein ZW10 in interphase cytoplasmic dynein function.

Zeste white 10 (ZW10) is a mitotic checkpoint protein and the anchor for cytoplasmic dynein at mitotic kinetochores, though it is expressed throughout the cell cycle. We find that ZW10 localizes to pericentriolar membranous structures during interphase and cosediments with Golgi membranes. Dominant-negative ZW10, anti-ZW10 antibody, and ZW10 RNA interference (RNAi) caused Golgi dispersal. ZW10 RNAi also dispersed endosomes and lysosomes. Live imaging of Golgi, endosomal, and lysosomal markers after reduced ZW10 expression showed a specific decrease in the frequency of minus end-directed movements. Golgi membrane-associated dynein was markedly decreased, suggesting a role for ZW10 in dynein cargo binding during interphase. We also find ZW10 enriched at the leading edge of migrating fibroblasts, suggesting that ZW10 serves as a general regulator of dynein function throughout the cell cycle.

Dual subcellular roles for LIS1 and dynein in radial neuronal migration in live brain tissue.

During brain development, neural precursor cells migrate along radial glial fibers to populate the neocortex. RNA interference (RNAi) of the lissencephaly gene LIS1 (also known as PAFAH1b1) inhibits somal movement but not process extension of neural precursors in live brain slices. Here we report imaging of the subcellular events accompanying neural precursor migration and the effects of LIS1, cytoplasmic dynein and myosin II inhibition. Centrosomes move continuously and often far in advance of nuclei, which show extreme saltatory behavior. LIS1 and dynein RNAi inhibit centrosomal and nuclear movement independently, whereas myosin II inhibition blocks only nuclear translocation. Imaging of the microtubule end-binding protein 3 (EB3) reveals a centrosome-centered array of microtubules in live neural precursors under all conditions examined. Dynein is concentrated both at a swelling in the leading process reported to initiate each migratory cycle and in the soma. Thus, dynein pulls on the microtubule network from the swelling. The nucleus is transported along the trailing microtubules by dynein assisted by myosin II.

Roles of the multivalent dynein adaptors BicD2 and RILP in neurons.

Cytoplasmic dynein is responsible for all forms of retrograde transport in neurons and other cells. Work over several years has led to the identification of a class of coiled-coil domain containing "adaptor" proteins that are responsible for expanding dynein's range of cargo interactions, as well as regulating dynein motor behavior. This brief review focuses first on the BicD family of adaptor proteins, which clearly serve to expand the number of dynein cargo interactions. RILP, another adaptor protein, also interacts with multiple proteins. Surprisingly, this is to mediate a series of steps within a common pathway, higher eukaryotic autophagy. These distinct features have important implications for understanding the full range of dynein adaptor functions.

Morphine increases brain levels of ferritin heavy chain leading to inhibition of CXCR4-mediated survival signaling in neurons.

This study focuses on the effect of mu-opioid receptor agonists on CXCR4 signaling in neurons and the mechanisms involved in regulation of neuronal CXCR4 by opiates. The data show that CXCR4 is negatively modulated by long-term morphine treatments both in vitro and in vivo; CXCR4 inhibition is caused by direct stimulation of mu-opioid receptors in neurons, leading to alterations of ligand-induced CXCR4 phosphorylation and upregulation of protein ferritin heavy chain (FHC), a negative intracellular regulator of CXCR4. Reduced coupling of CXCR4 to G-proteins was found in the brain of morphine-treated rats, primarily cortex and hippocampus. CXCR4-induced G alpha(i)/G betagamma activities were suppressed after 24 h treatment of cortical neurons with morphine or the selective mu-opioid agonist DAMGO (D-Ala2-N-Me-Phe(4)-glycol(5)-enkephalin), as shown by analysis of downstream targets of CXCR4 (i.e., cAMP, Akt, and ERK1/2). These agonists also prevented CXCL12-induced phosphorylation of CXCR4, indicating a deficit of CXCR4 activation in these conditions. Indeed, morphine (or DAMGO) inhibited prosurvival signaling in neurons. These effects are not attributable to a reduction in CXCR4 expression or surface levels but rather to upregulation of FHC by opioids. The crucial role of FHC in inhibition of neuronal CXCR4 was confirmed by in vitro and in vivo RNA interference studies. Overall, these findings suggest that opiates interfere with normal CXCR4 function in the brain. By this mechanism, opiates could reduce the neuroprotective functions of CXCR4 and exacerbate neuropathology in opiate abusers who are affected by neuroinflammatory/infectious disorders, including neuroAIDS.

How dynein helps the cell find its center: a servomechanical model.

Cytoplasmic dynein is the major minus-end-directed microtubule motor protein in interphase cells. In addition to its well-established roles in vesicular transport and chromosome dynamics, cytoplasmic dynein also associates with the cell cortex. From this site, it appears to pull on the cytoplasmic microtubule network, influencing mitotic spindle orientation, nuclear position and other aspects of cell polarity and organization. Recent evidence indicates that the cell has the remarkable ability to calculate is geometric center, and, with the help of dynein, to position the centrosome at this central site. Here, we outline models to account for this behavior.

LIS1 RNA interference blocks neural stem cell division, morphogenesis, and motility at multiple stages.

Mutations in the human LIS1 gene cause the smooth brain disease classical lissencephaly. To understand the underlying mechanisms, we conducted in situ live cell imaging analysis of LIS1 function throughout the entire radial migration pathway. In utero electroporation of LIS1 small interference RNA and short hairpin dominant negative LIS1 and dynactin cDNAs caused a dramatic accumulation of multipolar progenitor cells within the subventricular zone of embryonic rat brains. This effect resulted from a complete failure in progression from the multipolar to the migratory bipolar state, as revealed by time-lapse analysis of brain slices. Surprisingly, interkinetic nuclear oscillations in the radial glial progenitors were also abolished, as were cell divisions at the ventricular surface. Those few bipolar cells that reached the intermediate zone also exhibited a complete block in somal translocation, although, remarkably, process extension persisted. Finally, axonal growth also ceased. These results identify multiple distinct and novel roles for LIS1 in nucleokinesis and process dynamics and suggest that nuclear position controls neural progenitor cell division.