DCLK1 phosphorylates the microtubule‐associated protein MAP7D1 to promote axon elongation in cortical neurons

Doublecortin‐like kinase 1 (DCLK1) is a member of the neuronal microtubule‐associated doublecortin (DCX) family and functions in multiple stages of neural development including radial migration and axon growth of cortical neurons. DCLK1 is suggested to play the roles in part through its protein kinase activity, yet the kinase substrates of DCLK1 remain largely unknown. Here we have identified MAP7D1 (microtubule‐associated protein 7 domain containing 1) as a novel substrate of DCLK1 by using proteomic analysis. MAP7D1 is expressed in developing cortical neurons, and knockdown of MAP7D1 in layer 2/3 cortical neurons results in a significant impairment of callosal axon elongation, but not of radial migration, in corticogenesis. We have further defined the serine 315 (Ser 315) of MAP7D1 as a DCLK1‐induced phosphorylation site and shown that overexpression of a phosphomimetic MAP7D1 mutant in which Ser 315 is substituted with glutamic acid (MAP7D1 S315E), but not wild‐type MAP7D1, fully rescues the axon elongation defects in Dclk1 knockdown neurons. These data demonstrate that DCLK1 phosphorylates MAP7D1 on Ser 315 to facilitate axon elongation of cortical neurons. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419–437, 2017     

Dynein is required for polarized dendritic transport and uniform microtubule orientation in axons

Axons and dendrites differ in both microtubule organization and in the organelles and proteins they contain. Here we show that the microtubule motor dynein has a crucial role in polarized transport and in controlling the orientation of axonal microtubules in Drosophila melanogaster dendritic arborization (da) neurons. Changes in organelle distribution within the dendritic arbors of dynein mutant neurons correlate with a proximal shift in dendritic branch position. Dynein is also necessary for the dendrite-specific localization of Golgi outposts and the ion channel Pickpocket. Axonal microtubules are normally oriented uniformly plus-end-distal; however, without dynein, axons contain both plus- and minus-end distal microtubules. These data suggest that dynein is required for the distinguishing properties of the axon and dendrites: without dynein, dendritic organelles and proteins enter the axon and the axonal microtubules are no longer uniform in polarity.     

DCX, a new mediator of the JNK pathway

Mutations in the X-linked gene DCX result in lissencephaly in males, and abnormal neuronal positioning in females, suggesting a role for this gene product during neuronal migration. In spite of several known protein interactions, the involvement of DCX in a signaling pathway is still elusive. Here we demonstrate that DCX is a substrate of JNK and interacts with both c-Jun N-terminal kinase (JNK) and JNK interacting protein (JIP). The localization of this signaling module in the developing brain suggests its functionality in migrating neurons. The localization of DCX at neurite tips is determined by its interaction with JIP and by the interaction of the latter with kinesin. DCX is phosphorylated by JNK in growth cones. DCX mutated in sites phosphorylated by JNK affected neurite outgrowth, and the velocity and relative pause time of migrating neurons. We hypothesize that during neuronal migration, there is a need to regulate molecular motors that are working in the cell in opposite directions: kinesin (a plus-end directed molecular motor) versus dynein (a minus-end directed molecular motor).     

The DCX-domain tandems of doublecortin and doublecortin-like kinase

The doublecortin-like domains (DCX), which typically occur in tandem, are novel microtubule-binding modules. DCX tandems are found in doublecortin, a 360-residue protein expressed in migrating neurons; the doublecortin-like kinase (DCLK); the product of the RP1 gene that is responsible for a form of inherited blindness; and several other proteins. Mutations in the gene encoding doublecortin cause lissencephaly in males and the 'double-cortex syndrome' in females. We here report a solution structure of the N-terminal DCX domain of human doublecortin and a 1.5 A resolution crystal structure of the equivalent domain from human DCLK. Both show a stable, ubiquitin-like tertiary fold with distinct structural similarities to GTPase-binding domains. We also show that the C-terminal DCX domains of both proteins are only partially folded. In functional assays, the N-terminal DCX domain of doublecortin binds only to assembled microtubules, whereas the C-terminal domain binds to both microtubules and unpolymerized tubulin.     

Sequential dynein effectors regulate axonal autophagosome motility in a maturation-dependent pathway

Autophagy is a degradative pathway required to maintain homeostasis. Neuronal autophagosomes form constitutively at the axon terminal and mature via lysosomal fusion during dynein-mediated transport to the soma. How the dynein–autophagosome interaction is regulated is unknown. Here, we identify multiple dynein effectors on autophagosomes as they transit along the axons of primary neurons. In the distal axon, JIP1 initiates autophagosomal transport. Autophagosomes in the mid-axon require HAP1 and Huntingtin. We find that HAP1 is a dynein activator, binding the dynein–dynactin complex via canonical and noncanonical interactions. JIP3 is on most axonal autophagosomes, but specifically regulates the transport of mature autolysosomes. Inhibiting autophagosomal transport disrupts maturation, and inhibiting autophagosomal maturation perturbs the association and function of dynein effectors; thus, maturation and transport are tightly linked. These results reveal a novel maturation-based dynein effector handoff on neuronal autophagosomes that is key to motility, cargo degradation, and the maintenance of axonal health.     

JIP1 regulates the directionality of APP axonal transport by coordinating kinesin and dynein motors

Regulation of the opposing kinesin and dynein motors that drive axonal transport is essential to maintain neuronal homeostasis. Here, we examine coordination of motor activity by the scaffolding protein JNK-interacting protein 1 (JIP1), which we find is required for long-range anterograde and retrograde amyloid precursor protein (APP) motility in axons. We identify novel interactions between JIP1 and kinesin heavy chain (KHC) that relieve KHC autoinhibition, activating motor function in single molecule assays. The direct binding of the dynactin subunit p150^Glued to JIP1 competitively inhibits KHC activation in vitro and disrupts the transport of APP in neurons. Together, these experiments support a model whereby JIP1 coordinates APP transport by switching between anterograde and retrograde motile complexes. We find that mutations in the JNK-dependent phosphorylation site S421 in JIP1 alter both KHC activation in vitro and the directionality of APP transport in neurons. Thus phosphorylation of S421 of JIP1 serves as a molecular switch to regulate the direction of APP transport in neurons.     

CRMP/UNC-33 organizes microtubule bundles for KIF5-mediated mitochondrial distribution to axon

Neurons are highly specialized cells with polarized cellular processes and subcellular domains. As vital organelles for neuronal functions, mitochondria are distributed by microtubule-based transport systems. Although the essential components of mitochondrial transport including motors and cargo adaptors are identified, it is less clear how mitochondrial distribution among somato-dendritic and axonal compartment is regulated. Here, we systematically study mitochondrial motors, including four kinesins, KIF5, KIF17, KIF1, KLP-6, and dynein, and transport regulators in C. elegans PVD neurons. Among all these motors, we found that mitochondrial export from soma to neurites is mainly mediated by KIF5/UNC-116. Interestingly, UNC-116 is especially important for axonal mitochondria, while dynein removes mitochondria from all plus-end dendrites and the axon. We surprisingly found one mitochondrial transport regulator for minus-end dendritic compartment, TRAK-1, and two mitochondrial transport regulators for axonal compartment, CRMP/UNC-33 and JIP3/UNC-16. While JIP3/UNC-16 suppresses axonal mitochondria, CRMP/UNC-33 is critical for axonal mitochondria; nearly no axonal mitochondria present in unc-33 mutants. We showed that UNC-33 is essential for organizing the population of UNC-116-associated microtubule bundles, which are tracks for mitochondrial trafficking. Disarrangement of these tracks impedes mitochondrial transport to the axon. In summary, we identified a compartment-specific transport regulation of mitochondria by UNC-33 through organizing microtubule tracks for different kinesin motors other than microtubule polarity.     

PAR3 and aPKC regulate Golgi organization through CLASP2 phosphorylation to generate cell polarity

The organization of the Golgi apparatus is essential for cell polarization and its maintenance. The polarity regulator PAR complex (PAR3, PAR6, and aPKC) plays critical roles in several processes of cell polarization. However, how the PAR complex participates in regulating the organization of the Golgi remains largely unknown. Here we demonstrate the functional cross-talk of the PAR complex with CLASP2, which is a microtubule plus-end–tracking protein and is involved in organizing the Golgi ribbon. CLASP2 directly interacted with PAR3 and was phosphorylated by aPKC. In epithelial cells, knockdown of either PAR3 or aPKC induced the aberrant accumulation of CLASP2 at the trans-Golgi network (TGN) concomitantly with disruption of the Golgi ribbon organization. The expression of a CLASP2 mutant that inhibited the PAR3-CLASP2 interaction disrupted the organization of the Golgi ribbon. CLASP2 is known to localize to the TGN through its interaction with the TGN protein GCC185. This interaction was inhibited by the aPKC-mediated phosphorylation of CLASP2. Furthermore, the nonphosphorylatable mutant enhanced the colocalization of CLASP2 with GCC185, thereby perturbing the Golgi organization. On the basis of these observations, we propose that PAR3 and aPKC control the organization of the Golgi through CLASP2 phosphorylation.     

JIP3 Activates Kinesin-1 Motility to Promote Axon Elongation

Kinesin-1 is a molecular motor responsible for cargo transport along microtubules and plays critical roles in polarized cells, such as neurons. Kinesin-1 can function as a dimer of two kinesin heavy chains (KHC), which harbor the motor domain, or as a tetramer in combination with two accessory light chains (KLC). To ensure proper cargo distribution, kinesin-1 activity is precisely regulated. Both KLC and KHC subunits bind cargoes or regulatory proteins to engage the motor for movement along microtubules. We previously showed that the scaffolding protein JIP3 interacts directly with KHC in addition to its interaction with KLC and positively regulates dimeric KHC motility. Here we determined the stoichiometry of JIP3-KHC complexes and observed approximately four JIP3 molecules binding per KHC dimer. We then determined whether JIP3 activates tetrameric kinesin-1 motility. Using an in vitro motility assay, we show that JIP3 binding to KLC engages kinesin-1 with microtubules and that JIP3 binding to KHC promotes kinesin-1 motility along microtubules. We tested the in vivo relevance of these findings using axon elongation as a model for kinesin-1-dependent cellular function. We demonstrate that JIP3 binding to KHC, but not KLC, is essential for axon elongation in hippocampal neurons as well as axon regeneration in sensory neurons. These findings reveal that JIP3 regulation of kinesin-1 motility is critical for axon elongation and regeneration.     

An OBSL1-Cul7Fbxw8 ubiquitin ligase signaling mechanism regulates Golgi morphology and dendrite patterning

The elaboration of dendrites in neurons requires secretory trafficking through the Golgi apparatus, but the mechanisms that govern Golgi function in neuronal morphogenesis in the brain have remained largely unexplored. Here, we report that the E3 ubiquitin ligase Cul7(Fbxw8) localizes to the Golgi complex in mammalian brain neurons. Inhibition of Cul7(Fbxw8) by independent approaches including Fbxw8 knockdown reveals that Cul7(Fbxw8) is selectively required for the growth and elaboration of dendrites but not axons in primary neurons and in the developing rat cerebellum in vivo. Inhibition of Cul7(Fbxw8) also dramatically impairs the morphology of the Golgi complex, leading to deficient secretory trafficking in neurons. Using an immunoprecipitation/mass spectrometry screening approach, we also uncover the cytoskeletal adaptor protein OBSL1 as a critical regulator of Cul7(Fbxw8) in Golgi morphogenesis and dendrite elaboration. OBSL1 forms a physical complex with the scaffold protein Cul7 and thereby localizes Cul7 at the Golgi apparatus. Accordingly, OBSL1 is required for the morphogenesis of the Golgi apparatus and the elaboration of dendrites. Finally, we identify the Golgi protein Grasp65 as a novel and physiologically relevant substrate of Cul7(Fbxw8) in the control of Golgi and dendrite morphogenesis in neurons. Collectively, these findings define a novel OBSL1-regulated Cul7(Fbxw8) ubiquitin signaling mechanism that orchestrates the morphogenesis of the Golgi apparatus and patterning of dendrites, with fundamental implications for our understanding of brain development.     

Neuronal polarity in Drosophila: sorting out axons and dendrites

Drosophila neurons have identifiable axons and dendrites based on cell shape, but it is only just starting to become clear how Drosophila neurons are polarized at the molecular level. Dendrite-specific components including the Golgi complex, GABA receptors, neurotransmitter receptor scaffolding proteins, and cell adhesion molecules have been described. Proteins involved in constructing presynaptic specializations are concentrated in axons of some neurons. A very simple model for how these components are distributed to axons and dendrites can be constructed based on the opposite polarity of microtubules in axons and dendrites: dynein carries cargo into dendrites, and kinesins carry cargo into axons. The simple model works well for multipolar neurons, but will likely need refinement for unipolar neurons, which are common in Drosophila.     

Microtubule nucleation at the cis‐side of the Golgi apparatus requires AKAP450 and GM130

We report that microtubule (MT) nucleation at the Golgi apparatus requires AKAP450, a centrosomal γ‐TuRC‐interacting protein that also forms a distinct network associated with the Golgi. Depletion of AKAP450 abolished MT nucleation at the Golgi, whereas depletion of the cis‐Golgi protein GM130 led to the disorganisation of AKAP450 network and impairment of MT nucleation. Brefeldin‐A treatment induced relocalisation of AKAP450 to ER exit sites and concomitant redistribution of MT nucleation capacity to the ER. AKAP450 specifically binds the cis‐side of the Golgi in an MT‐independent, GM130‐dependent manner. Short AKAP450‐dependent growing MTs are covered by CLASP2. Like for centrosome, dynein/dynactin complexes are necessary to anchor MTs growing from the Golgi. We further show that Golgi‐associated AKAP450 has a role in cell migration rather than in cell polarisation of the centrosome–Golgi apparatus. We propose that the recruitment of AKAP450 on the Golgi membranes through GM130 allows centrosome‐associated nucleating activity to extend to the Golgi, to control the assembly of subsets of MTs ensuring specific functions within the Golgi or for transporting specific cargos to the cell periphery.     

Golgi Positioning

The Golgi apparatus in mammalian cells is positioned near the centrosome-based microtubule-organizing center (Fig. 1). Secretory cargo moves inward in membrane carriers for delivery to Golgi membranes in which it is processed and packaged for transport outward to the plasma membrane. Cytoplasmic dynein motor proteins (herein termed dynein) primarily mediate inward cargo carrier movement and Golgi positioning. These motors move along microtubules toward microtubule minus-ends embedded in centrosomes. Centripetal motility is controlled by a host of regulators whose precise functions remain to be determined. Significantly, a specific Golgi receptor for dynein has not been identified. This has impaired progress toward elucidation of membrane-motor-microtubule attachment in the periphery and, after inward movement, recycling of the motor for another round. Pericentrosomal positioning of the Golgi apparatus is dynamic. It is regulated during critical cellular processes such as mitosis, differentiation, cell polarization, and cell migration. Positioning is also important as it aligns the Golgi along an axis of cell polarity. In certain cell types, this promotes secretion directed to the proximal plasma membrane domain thereby maintaining specializations critical for diverse processes including wound healing, immunological synapse formation, and axon determination.     

Doublecortin-like kinase controls neurogenesis by regulating mitotic spindles and M phase progression

The mechanisms controlling neurogenesis during brain development remain relatively unknown. Through a differential protein screen with developmental versus mature neural tissues, we identified a group of developmentally enriched microtubule-associated proteins (MAPs) including doublecortin-like kinase (DCLK), a protein that shares high homology with doublecortin (DCX). DCLK, but not DCX, is highly expressed in regions of active neurogenesis in the neocortex and cerebellum. Through a dynein-dependent mechanism, DCLK regulates the formation of bipolar mitotic spindles and the proper transition from prometaphase to metaphase during mitosis. In cultured cortical neural progenitors, DCLK RNAi Lentivirus disrupts the structure of mitotic spindles and the progression of M phase, causing an increase of cell-cycle exit index and an ectopic commitment to a neuronal fate. Furthermore, both DCLK gain and loss of function in vivo specifically promote a neuronal identity in neural progenitors. These data provide evidence that DCLK controls mitotic division by regulating spindle formation and also determines the fate of neural progenitors during cortical neurogenesis.     

Lrrk regulates the dynamic profile of dendritic Golgi outposts through the golgin Lava lamp

Constructing the dendritic arbor of neurons requires dynamic movements of Golgi outposts (GOPs), the prominent component in the dendritic secretory pathway. GOPs move toward dendritic ends (anterograde) or cell bodies (retrograde), whereas most of them remain stationary. Here, we show that Leucine-rich repeat kinase (Lrrk), the Drosophila melanogaster homologue of Parkinson’s disease–associated Lrrk2, regulates GOP dynamics in dendrites. Lrrk localized at stationary GOPs in dendrites and suppressed GOP movement. In Lrrk loss-of-function mutants, anterograde movement of GOPs was enhanced, whereas Lrrk overexpression increased the pool size of stationary GOPs. Lrrk interacted with the golgin Lava lamp and inhibited the interaction between Lva and dynein heavy chain, thus disrupting the recruitment of dynein to Golgi membranes. Whereas overexpression of kinase-dead Lrrk caused dominant-negative effects on GOP dynamics, overexpression of the human LRRK2 mutant G2019S with augmented kinase activity promoted retrograde movement. Our study reveals a pathogenic pathway for LRRK2 mutations causing dendrite degeneration.     

Growing dendrites and axons differ in their reliance on the secretory pathway

Little is known about how the distinct architectures of dendrites and axons are established. From a genetic screen, we isolated dendritic arbor reduction (dar) mutants with reduced dendritic arbors but normal axons of Drosophila neurons. We identified dar2, dar3, and dar6 genes as the homologs of Sec23, Sar1, and Rab1 of the secretory pathway. In both Drosophila and rodent neurons, defects in Sar1 expression preferentially affected dendritic growth, revealing evolutionarily conserved difference between dendritic and axonal development in the sensitivity to limiting membrane supply from the secretory pathway. Whereas limiting ER-to-Golgi transport resulted in decreased membrane supply from soma to dendrites, membrane supply to axons remained sustained. We also show that dendritic growth is contributed by Golgi outposts, which are found predominantly in dendrites. The distinct dependence between dendritic and axonal growth on the secretory pathway helps to establish different morphology of dendrites and axons.     

Interaction of early secretory pathway and Golgi membranes with microtubules and microtubule motors

This review summarizes the data describing the role of cellular microtubules in transportation of membrane vesicles — transport containers for secreted proteins or lipids. Most events of early vesicular transport in animal cells (from the endoplasmic reticulum to the Golgi apparatus and in the opposite recycling direction) are mediated by microtubules and microtubule motor proteins. Data on the role of dynein and kinesin in early vesicle transport remain controversial, probably because of the differentiated role of these proteins in the movements of vesicles or membrane tubules with various cargos and at different stages of secretion and retrograde transport. Microtubules and dynein motor protein are essential for maintaining a compact structure of the Golgi apparatus; moreover, there is a set of proteins that are essential for Golgi compactness. Dispersion of ribbon-like Golgi often occurs under physiological conditions in interphase cells. Golgi is localized in the leading part of crawling cultured fibroblasts, which also depends on microtubules and dynein. The Golgi apparatus creates its own system of microtubules by attracting γ-tubulin and some microtubule-associated proteins to membranes. Molecular mechanisms of binding microtubule-associated and motor proteins to membranes are very diverse, suggesting the possibility of regulation of Golgi interaction with microtubules during cell differentiation. To illustrate some statements, we present our own data showing that the cluster of vesicles induced by expression of constitutively active GTPase Sar1a[H79G] in cells is dispersed throughout the cell after microtubule disruption. Movement of vesicles in cells containing the intermediate compartment protein ERGIC53/LMANI was inhibited by inhibiting dynein. Inhibiting protein kinase LOSK/SLK prevented orientation of Golgi to the leading part of crawling cells, but the activity of dynein was not inhibited according to data on the movement of ERGIC53/LMANI-marked vesicles.     

Cytoplasmic dynein participates in the centrosomal localization of the Golgi complex

The localization of the Golgi complex depends upon the integrity of the microtubule apparatus. At interphase, the Golgi has a restricted pericentriolar localization. During mitosis, it fragments into small vesicles that are dispersed throughout the cytoplasm until telophase, when they again coalesce near the centrosome. These observations have suggested that the Golgi complex utilizes a dynein-like motor to mediate its transport from the cell periphery towards the minus ends of microtubules, located at the centrosome. We utilized semi-intact cells to study the interaction of the Golgi complex with the microtubule apparatus. We show here that Golgi complexes can enter semi-intact cells and associate stably with cytoplasmic constituents. Stable association, termed here "Golgi capture," requires ATP hydrolysis and intact microtubules, and occurs maximally at physiological temperature in the presence of added cytosolic proteins. Once translocated into the semi-intact cell cytoplasm, exogenous Golgi complexes display a distribution similar to endogenous Golgi complexes, near the microtubule-organizing center. The process of Golgi capture requires cytoplasmic tubulin, and is abolished if cytoplasmic dynein is immunodepleted from the cytosol. Cytoplasmic dynein, prepared from CHO cell cytosol, restores Golgi capture activity to reactions carried out with dynein immuno-depleted cytosol. These results indicate that cytoplasmic dynein can interact with isolated Golgi complexes, and participate in their accumulation near the centrosomes of semi-intact, recipient cells. Thus, cytoplasmic dynein appears to play a role in determining the subcellular localization of the Golgi complex.     

Mon1a protein acts in trafficking through the secretory apparatus

Mon1a was originally identified as a modifier gene of vesicular traffic, as a mutant Mon1a allele resulted in increased localization of cell surface proteins, whereas reduced levels of Mon1a showed decreased secretory activity. Here we show that Mon1a affects different steps in the secretory pathway including endoplasmic reticulum-to-Golgi traffic. siRNA-dependent reduction of Mon1a levels resulted in a delay in the reformation of the Golgi apparatus after Brefeldin A treatment. Endoglycosidase H treatment of ts045VSVG-GFP confirmed that knockdown of Mon1a delayed endoplasmic reticulum-to-Golgi trafficking. Reductions in Mon1a also resulted in delayed trafficking from Golgi to the plasma membrane. Immunoprecipitation and mass spectrometry analysis showed that Mon1a associates with dynein intermediate chain. Reductions in Mon1a or dynein altered steady state Golgi morphology. Reductions in Mon1a delayed formation of ERGIC-53-positive vesicles, whereas reductions in dynein did not affect vesicle formation. These data provide strong evidence for a role for Mon1a in anterograde trafficking through the secretory apparatus.     

Microtubule‐binding protein doublecortin‐like kinase 1 (DCLK1) guides kinesin‐3‐mediated cargo transport to dendrites

In neurons, the polarized distribution of vesicles and other cellular materials is established through molecular motors that steer selective transport between axons and dendrites. It is currently unclear whether interactions between kinesin motors and microtubule‐binding proteins can steer polarized transport. By screening all 45 kinesin family members, we systematically addressed which kinesin motors can translocate cargo in living cells and drive polarized transport in hippocampal neurons. While the majority of kinesin motors transport cargo selectively into axons, we identified five members of the kinesin‐3 (KIF1) and kinesin‐4 (KIF21) subfamily that can also target dendrites. We found that microtubule‐binding protein doublecortin‐like kinase 1 (DCLK1) labels a subset of dendritic microtubules and is required for KIF1‐dependent dense‐core vesicles (DCVs) trafficking into dendrites and dendrite development. Our study demonstrates that microtubule‐binding proteins can provide local signals for specific kinesin motors to drive polarized cargo transport.