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.

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.

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.

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.

Nesprin-2 Recruitment of BicD2 to the Nuclear Envelope Controls Dynein/Kinesin-Mediated Neuronal Migration In Vivo.

Vertebrate brain development depends on a complex program of cell proliferation and migration. Post-mitotic neuronal migration in the developing cerebral cortex involves Nesprin-2, which recruits cytoplasmic dynein, kinesin, and actin to the nuclear envelope (NE) in other cell types. However, the relative importance of these interactions in neurons has remained poorly understood. To address these issues, we performed in utero electroporation into the developing rat brain to interfere with Nesprin-2 function. We find that an ∼100-kDa "mini" form of the ∼800-kDa Nesprin-2 protein, which binds dynein and kinesin, is sufficient, remarkably, to support neuronal migration. In contrast to dynein's role in forward nuclear migration in these cells, we find that kinesin-1 inhibition accelerates neuronal migration, suggesting a novel role for the opposite-directed motor proteins in regulating migration velocity. In contrast to studies in fibroblasts, the actin-binding domain of Nesprin-2 was dispensable for neuronal migration. We find further that, surprisingly, the motor proteins interact with Nesprin-2 through the dynein/kinesin "adaptor" BicD2, both in neurons and in non-mitotic fibroblasts. Furthermore, mutation of the Nesprin-2 LEWD sequence, implicated in nuclear envelope kinesin recruitment in other systems, interferes with BicD2 binding. Although disruption of the Nesprin-2/BicD2 interaction severely inhibited nuclear movement, centrosome advance proceeded unimpeded, supporting an independent mechanism for centrosome advance. Our data together implicate Nesprin-2 as a novel and fundamentally important form of BicD2 cargo and help explain BicD2's role in neuronal migration and human disease.

A RILP-regulated pathway coordinating autophagosome biogenesis with transport.

Mammalian cells, including neurons, use macroautophagy (here 'autophagy') to degrade damaged proteins and organelles, and recycle nutrients in response to starvation and other forms of cell stress. The basic cellular machinery responsible for autophagy is highly conserved from yeast to mammals. However, evidence for specific adaptations to more complex organisms and in highly differentiated cells (e. g. neurons) remains limited. RILP (Rab interacting lysosomal protein) mediates retrograde transport of late endosomes (LEs) in nonneuronal mammalian cells. We have now found that RILP plays additional important, fundamental roles in neuronal autophagosome (AP) transport, and, more surprisingly, in AP biogenesis, and cargo turnover as well. RILP accomplishes these tasks via sequential interactions with key autophagosomal components - ATG5 and LC3 - as well as the microtubule motor protein cytoplasmic dynein (Figure 1A). We found further that RILP expression and behavior are controlled by MTOR kinase, linking RILP to a potentially wide range of physiological and pathophysiological functions.

The Dynein Adaptor RILP Controls Neuronal Autophagosome Biogenesis, Transport, and Clearance.

Autophagy plays critical roles in neurodegeneration and development, but how this pathway is organized and regulated in neurons remains poorly understood. Here, we find that the dynein adaptor RILP is essential for retrograde transport of neuronal autophagosomes, and surprisingly, their biogenesis as well. We find that induction of autophagy by mTOR inhibition specifically upregulates RILP expression and its localization to autophagosomes. RILP depletion or mutations in its LC3-binding LIR motifs strongly decrease autophagosome numbers suggesting an unexpected RILP role in autophagosome biogenesis. We find that RILP also interacts with ATG5 on isolation membranes, precluding premature dynein recruitment and autophagosome transport. RILP inhibition impedes autophagic turnover and causes p62/sequestosome-1 aggregation. Together, our results identify an mTOR-responsive neuronal autophagy pathway, wherein RILP integrates the processes of autophagosome biogenesis and retrograde transport to control autophagic turnover. This pathway has important implications for understanding how autophagy contributes to neuronal function, development, and disease.

Role of cytoplasmic dynein and kinesins in adenovirus transport.

Following receptor-mediated uptake into endocytic vesicles and subsequent escape, adenovirus particles are transported along microtubules. The microtubule motor proteins dynein and one or more kinesins are involved in this behavior. Dynein is implicated in adenovirus transport toward the nucleus. The kinesin Kif5B has now been found to move the adenovirus (AdV) toward microtubule plus ends, though a kinesin role in adenovirus-induced nuclear pore disruption has also been reported. In undifferentiated cells, dynein-mediated transport predominates early in infection, but motility becomes bidirectional with time. The latter behavior can be modeled as a novel assisted diffusion mechanism, which may allow virus particles to explore the cytoplasm more efficiently. Cytoplasmic dynein and Kif5B have both been found to bind AdV through direct interactions with the capsid proteins hexon and penton base, respectively. We review here the roles of the microtubule motor proteins in AdV infection, the relationship between motor protein recruitment to pathogenic vs. physiological cargoes, the evolutionary origins of microtubule-mediated AdV transport, and a role for the motor proteins in a novel host-defense mechanism.

Distinct roles for dynein light intermediate chains in neurogenesis, migration, and terminal somal translocation.

Cytoplasmic dynein participates in multiple aspects of neocortical development. These include neural progenitor proliferation, morphogenesis, and neuronal migration. The cytoplasmic dynein light intermediate chains (LICs) 1 and 2 are cargo-binding subunits, though their relative roles are not well understood. Here, we used in utero electroporation of shRNAs or LIC functional domains to determine the relative contributions of the two LICs in the developing rat brain. We find that LIC1, through BicD2, is required for apical nuclear migration in neural progenitors. In newborn neurons, we observe specific roles for LIC1 in the multipolar to bipolar transition and glial-guided neuronal migration. In contrast, LIC2 contributes to a novel dynein role in the little-studied mode of migration, terminal somal translocation. Together, our results provide novel insight into the LICs' unique functions during brain development and dynein regulation overall.

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.

Cdk1 phosphorylation of the dynein adapter Nde1 controls cargo binding from G2 to anaphase.

Cytoplasmic dynein is involved in diverse cell cycle-dependent functions regulated by several accessory factors, including Nde1 and Ndel1. Little is known about the role of these proteins in dynein cargo binding, and less is known about their cell cycle--dependent dynein regulation. Using Nde1 RNAi, mutant cDNAs, and a phosphorylation site-specific antibody, we found a specific association of phospho-Nde1 with the late G2-M nuclear envelope and prophase to anaphase kinetochores, comparable to the pattern for the Nde1 interactor CENP-F. Phosphomutant-Nde1 associated only with prometaphase kinetochores and showed weaker CENP-F binding in in vitro assays. Nde1 RNAi caused severe delays in mitotic progression, which were substantially rescued by both phosphomimetic and phosphomutant Nde1. Expression of a dynein-binding-deficient Nde1 mutant reduced kinetochore dynein by half, indicating a major role for Nde1 in kinetochore dynein recruitment. These results establish CENP-F as the first well-characterized Nde1 cargo protein, and reveal phosphorylation control of Nde1 cargo binding throughout a substantial fraction of the cell cycle.

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.

Load-induced enhancement of Dynein force production by LIS1-NudE in vivo and in vitro.

Most sub-cellular cargos are transported along microtubules by kinesin and dynein molecular motors, but how transport is regulated is not well understood. It is unknown whether local control is possible, for example, by changes in specific cargo-associated motor behaviour to react to impediments. Here we discover that microtubule-associated lipid droplets (LDs) in COS1 cells respond to an optical trap with a remarkable enhancement in sustained force production. This effect is observed only for microtubule minus-end-moving LDs. It is specifically blocked by RNAi for the cytoplasmic dynein regulators LIS1 and NudE/L (Nde1/Ndel1), but not for the dynactin p150(Glued) subunit. It can be completely replicated using cell-free preparations of purified LDs, where duration of LD force production is more than doubled. These results identify a novel, intrinsic, cargo-associated mechanism for dynein-mediated force adaptation, which should markedly improve the ability of motor-driven cargoes to overcome subcellular obstacles.

Severe NDE1-mediated microcephaly results from neural progenitor cell cycle arrests at multiple specific stages.

Microcephaly is a cortical malformation disorder characterized by an abnormally small brain. Recent studies have revealed severe cases of microcephaly resulting from human mutations in the NDE1 gene, which is involved in the regulation of cytoplasmic dynein. Here using in utero electroporation of NDE1 short hairpin RNA (shRNA) in embryonic rat brains, we observe cell cycle arrest of proliferating neural progenitors at three distinct stages: during apical interkinetic nuclear migration, at the G2-to-M transition and in regulation of primary cilia at the G1-to-S transition. RNAi against the NDE1 paralogue NDEL1 has no such effects. However, NDEL1 overexpression can functionally compensate for NDE1, except at the G2-to-M transition, revealing a unique NDE1 role. In contrast, NDE1 and NDEL1 RNAi have comparable effects on postmitotic neuronal migration. These results reveal that the severity of NDE1-associated microcephaly results not from defects in mitosis, but rather the inability of neural progenitors to ever reach this stage.

Emerging roles for motor proteins in progenitor cell behavior and neuronal migration during brain development.

Over the past two decades, substantial progress has been made in visualizing and understanding neuronal cell migration and morphogenesis during brain development. Distinct mechanisms have evolved to support migration of the various cell types that compose the developing neocortex. A specific subset of molecular motors, so far consisting of cytoplasmic dynein 1, Kif1a and myosin II, are responsible for cytoskeletal and nuclear transport in these cells. This review focuses on the emerging roles for each of these motor proteins in the migratory mechanisms of neocortical cell types. We discuss how migration can be cell cycle regulated and how coordination of motor activity is required to ensure migratory direction. © 2016 Wiley Periodicals, Inc.

KIF1A inhibition immortalizes brain stem cells but blocks BDNF-mediated neuronal migration.

Brain neural stem cells (radial glial progenitors, RGPs) undergo a mysterious form of cell cycle-entrained interkinetic nuclear migration (INM) that is driven apically by cytoplasmic dynein and basally by the kinesin KIF1A, which has recently been implicated in human brain developmental disease. To understand the consequences of altered basal INM and the roles of KIF1A in disease, we performed constitutive and conditional RNAi and expressed mutant KIF1A in E16 to P7 rat RGPs and neurons. RGPs inhibited in basal INM still showed normal cell cycle progression, although neurogenic divisions were severely reduced. Postmitotic neuronal migration was independently disrupted at the multipolar stage and accompanied by premature ectopic expression of neuronal differentiation markers. Similar effects were unexpectedly observed throughout the layer of surrounding control cells, mimicked by Bdnf (brain-derived neurotrophic factor) or Dcx RNAi, and rescued by BDNF application. These results identify sequential and independent roles for KIF1A and provide an important new approach for reversing the effects of human disease.

Cellular and subcellular imaging of motor protein-based behavior in embryonic rat brain.

Development of the cerebral cortex is a very dynamic process, involving a series of complex morphogenetic events. Following division of progenitor cells in the ventricular zone, neurons undergo a series of morphological changes and migrate outward toward the cortical plate, where they differentiate and integrate into functional circuits. Errors at several of stages during neurogenesis and migration cause a variety of severe cortical malformations. A number of disease genes encode factors associated with the cytoskeleton, which plays a crucial role throughout cortical development. Methods for regulating gene expression coupled with imaging of subcellular structures have provided important insight into the mechanisms governing normal and abnormal brain development. We describe here a series of protocols for imaging motor protein-dependent processes in real time in the developing rat brain.

Imaging of motor-dependent transport in neuronal and nonneuronal cells at high spatial and temporal resolution.

A wide range of subcellular organelles, pathogens, and macromolecular complexes are actively transported within neuronal and nonneuronal cells by microtubule motors. Transport speeds range up to 2-3 µm/s, which requires millisecond- and nanometer-scale resolution for proper imaging and analysis. Dissecting the contributions of multiple motor types has been challenging because of their functional interdependence and the complexity of individual motor behavior. In this chapter, we describe several methods for motor inhibition coupled with high-resolution particle tracking of vesicular and virus cargoes to provide a detailed and quantitative understanding of motor behavior and regulation. We discuss long-term inhibition, as well as short-term inhibition methods when needed to minimize complications from motor protein interactions.