Disruption of Axonal Transport Perturbs Bone Morphogenetic Protein (BMP) - Signaling and Contributes to Synaptic Abnormalities in Two Neurodegenerative Diseases

Formation of new synapses or maintenance of existing synapses requires the delivery of synaptic components from the soma to the nerve termini via axonal transport. One pathway that is important in synapse formation, maintenance and function of the Drosophila neuromuscular junction (NMJ) is the bone morphogenetic protein (BMP)-signaling pathway. Here we show that perturbations in axonal transport directly disrupt BMP signaling, as measured by its downstream signal, phospho Mad (p-Mad). We found that components of the BMP pathway genetically interact with both kinesin-1 and dynein motor proteins. Thick vein (TKV) vesicle motility was also perturbed by reductions in kinesin-1 or dynein motors. Interestingly, dynein mutations severely disrupted p-Mad signaling while kinesin-1 mutants showed a mild reduction in p-Mad signal intensity. Similar to mutants in components of the BMP pathway, both kinesin-1 and dynein motor protein mutants also showed synaptic morphological defects. Strikingly TKV motility and p-Mad signaling were disrupted in larvae expressing two human disease proteins; expansions of glutamine repeats (polyQ77) and human amyloid precursor protein (APP) with a familial Alzheimer''s disease (AD) mutation (APPswe). Consistent with axonal transport defects, larvae expressing these disease proteins showed accumulations of synaptic proteins along axons and synaptic abnormalities. Taken together our results suggest that similar to the NGF-TrkA signaling endosome, a BMP signaling endosome that directly interacts with molecular motors likely exist. Thus problems in axonal transport occurs early, perturbs BMP signaling, and likely contributes to the synaptic abnormalities observed in these two diseases.     

Dynactin enhances the processivity of kinesin-2

Kinesin-2 is a major microtubule-based motor in most cell types. Its in vitro motile properties have been analyzed extensively and been found to differ considerably from kinesin-1. Although recombinant kinesin-2 heterodimers exhibit processive movement, the processivity of the native kinesin-2 holoenzyme has never been evaluated. Kinesin-2 can interact with dynactin, a 'processivity factor' for cytoplasmic dynein, which may alter its motile properties. In this study, we analyze the in vitro motility of single native kinesin-2 molecules and determine the effects of dynactin on motor processivity. We find that individual native kinesin-2 molecules travel processively. Dynactin has no effect on velocity but significantly increases the run length of kinesin-2 movements. These results show that the interaction with dynactin has important functional consequences on the activity of the kinesin-2 motor.     

Molecular analysis of the klarsicht gene and its role in nuclear migration within differentiating cells of the Drosophila eye.

BACKGROUND: The temporally regulated, cell-type-specific transport of organelles has great biological significance, yet little is known about the regulation of organelle transport during development. The Drosophila gene klarsicht is required for temporally regulated lipid droplet transport in developing embryos and for the stereotypical nuclear migrations in differentiating cells of the developing eye. Klarsicht is thought to coordinate the function of several molecular motors bound to a single lipid droplet or to facilitate the attachment of dynein to the cargo, but it is not known whether Klarsicht affects motors directly or indirectly. RESULTS: Here, we have cloned the klarsicht gene and shown that it encodes a unique large protein. Drosophila klarsicht null mutants were viable, with obvious defects only in adult eye morphology. Epitope-tagged Klarsicht expressed in the eye from a transgene was perinuclear. In flies carrying transgenes that express markers for microtubule plus and minus ends, microtubules in differentiating cells of the eye were oriented with their plus ends apical and their minus ends at the nucleus. CONCLUSIONS: Drosophila klarsicht null mutants were viable and fertile, demonstrating that klarsicht is essential only for specific motor protein functions. Perinuclear localization of Klarsicht protein indicates that Klarsicht has a direct mechanical role in nuclear migration. Taken together with the finding that the minus ends of the microtubules are associated with the photoreceptor nuclei, the observation that Klarsicht is largely perinuclear supports the idea that Klarsicht associates with dynein, consistent with a model in which Klarsicht assists dynein in 'reeling in' the nucleus.     

Regulatory ATPase sites of cytoplasmic dynein affect processivity and force generation

The heavy chain of cytoplasmic dynein contains four nucleotide-binding domains referred to as AAA1-AAA4, with the first domain (AAA1) being the main ATP hydrolytic site. Although previous studies have proposed regulatory roles for AAA3 and AAA4, the role of ATP hydrolysis at these sites remains elusive. Here, we have analyzed the single molecule motility properties of yeast cytoplasmic dynein mutants bearing mutations that prevent ATP hydrolysis at AAA3 or AAA4. Both mutants remain processive, but the AAA4 mutant exhibits a surprising increase in processivity due to its tighter affinity for microtubules. In addition to changes in motility characteristics, AAA3 and AAA4 mutants produce less maximal force than wild-type dynein. These results indicate that the nucleotide binding state at AAA3 and AAA4 can allosterically modulate microtubule binding affinity and affect dynein processivity and force production.     

A dynein loading zone for retrograde endosome motility at microtubule plus-ends

In the fungus Ustilago maydis, early endosomes move bidirectionally along microtubules (MTs) and facilitate growth by local membrane recycling at the tip of the infectious hypha. Here, we set out to elucidate the molecular mechanism of this process. We show that endosomes travel by Kinesin-3 activity into the hyphal apex, where they reverse direction and move backwards in a dynein-dependent manner. Our data demonstrate that dynein, dynactin and Lis1 accumulate at MT plus-ends within the hyphal tip, where they provide a reservoir of inactive motors for retrograde endosome transport. Consistently, endosome traffic is abolished after depletion of the dynein activator Lis1 and in Kinesin-1 null mutants, which was due to a defect in targeting of dynein and dynactin to the apical MT plus-ends. Furthermore, biologically active GFP-dynein travels on endosomes in retrograde and not in anterograde direction. Surprisingly, a CLIP170 homologue was neither needed for dynein localization nor for endosome transport. These results suggest an apical dynein loading zone in the hyphal tip, which ensure that endosomes reach the expanding growth region before they reverse direction.     

Integrins regulate DLG/FAS2 via a CaM kinase II-dependent pathway to mediate synapse elaboration and stabilization during postembryonic development

Calcium/calmodulin dependent kinase II (CaMKII), PDZ-domain scaffolding protein Discs-large (DLG), immunoglobin superfamily cell adhesion molecule Fasciclin 2 (FAS2) and the position specific (PS) integrin receptors, including betaPS and its alpha partners (alphaPS1, alphaPS2, alphaPS3/alphaVolado), are all known to regulate the postembryonic development of synaptic terminal arborization at the Drosophila neuromuscular junction (NMJ). Recent work has shown that DLG and FAS2 function together to modulate activity-dependent synaptic development and that this role is regulated by activation of CaMKII. We show that PS integrins function upstream of CaMKII in the development of synaptic architecture at the NMJ. betaPS integrin physically associates with the synaptic complex anchored by the DLG scaffolding protein, which contains CaMKII and FAS2. We demonstrate an alteration of the FAS2 molecular cascade in integrin regulatory mutants, as a result of CaMKII/integrin interactions. Regulatory betaPS integrin mutations increase the expression and synaptic localization of FAS2. Synaptic structural defects in betaPS integrin mutants are rescued by transgenic overexpression of CaMKII (proximal in pathway) or genetic reduction of FAS2 (distal in pathway). These studies demonstrate that betaPS integrins act through CaMKII activation to control the localization of synaptic proteins involved in the development of NMJ synaptic morphology.     

APLIP1, a kinesin binding JIP-1/JNK scaffold protein, influences the axonal transport of both vesicles and mitochondria in Drosophila

In a genetic screen for Kinesin heavy chain (Khc)-interacting proteins, we identified APLIP1, a neuronally expressed Drosophila homolog of JIP-1, a JNK scaffolding protein . JIP-1 and its homologs have been proposed to act as physical linkers between kinesin-1, which is a plus-end-directed microtubule motor, and certain anterograde vesicles in the axons of cultured neurons . Mutation of Aplip1 caused larval paralysis, axonal swellings, and reduced levels of both anterograde and retrograde vesicle transport, similar to the effects of kinesin-1 inhibition. In contrast, Aplip1 mutation caused a decrease only in retrograde transport of mitochondria, suggesting inhibition of the minus-end microtubule motor cytoplasmic dynein . Consistent with dynein defects, combining heterozygous mutations in Aplip1 and Dynein heavy chain (Dhc64C) generated synthetic axonal transport phenotypes. Thus, APLIP1 may be an important part of motor-cargo linkage complexes for both kinesin-1 and dynein. However, it is also worth considering that APLIP1 and its associated JNK signaling proteins could serve as an important signaling module for regulating transport by the two opposing motors.     

Modelling active transport in Drosophila unipolar motor neurons

This paper develops a model for simulating organelle transport in Drosophila unipolar motor neurons. The paper is motivated by a recent experimental investigation by Stone et al. (Microtubules have opposite orientation in axons and dendrites of Drosophila neurons. Mol Biol Cell.19:4122-4129) who proposed a map of microtubule (MT) orientation in Drosophila neurons, and explained why dynein mutations selectively impede dendritic growth without having much effect on axonal growth. Two different approaches to modelling the effect of dynein mutations are utilised: one through assuming a reduced average velocity of a dynein mutant motor and the other through assuming its decreased processivity (an increased detachment rate from MTs). Modified Smith–Simmons equations are used for developing a continuum model of the process. Distributions of organelle concentrations as well as distributions of diffusion, motor-driven and total organelle fluxes are simulated.     

Modelling active transport in Drosophila unipolar motor neurons

This paper develops a model for simulating organelle transport in Drosophila unipolar motor neurons. The paper is motivated by a recent experimental investigation by Stone et al. (Microtubules have opposite orientation in axons and dendrites of Drosophila neurons. Mol Biol Cell.19:4122-4129) who proposed a map of microtubule (MT) orientation in Drosophila neurons, and explained why dynein mutations selectively impede dendritic growth without having much effect on axonal growth. Two different approaches to modelling the effect of dynein mutations are utilised: one through assuming a reduced average velocity of a dynein mutant motor and the other through assuming its decreased processivity (an increased detachment rate from MTs). Modified Smith-Simmons equations are used for developing a continuum model of the process. Distributions of organelle concentrations as well as distributions of diffusion, motor-driven and total organelle fluxes are simulated.     

Drosophila roadblock and Chlamydomonas LC7: a conserved family of dynein-associated proteins involved in axonal transport, flagellar motility, and mitosis.

Eukaryotic organisms utilize microtubule-dependent motors of the kinesin and dynein superfamilies to generate intracellular movement. To identify new genes involved in the regulation of axonal transport in Drosophila melanogaster, we undertook a screen based upon the sluggish larval phenotype of known motor mutants. One of the mutants identified in this screen, roadblock (robl), exhibits diverse defects in intracellular transport including axonal transport and mitosis. These defects include intra-axonal accumulations of cargoes, severe axonal degeneration, and aberrant chromosome segregation. The gene identified by robl encodes a 97-amino acid polypeptide that is 57% identical (70% similar) to the 105-amino acid Chlamydomonas outer arm dynein-associated protein LC7, also reported here. Both robl and LC7 have homology to several other genes from fruit fly, nematode, and mammals, but not Saccharomyces cerevisiae. Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein. We propose that roadblock/LC7 family members may modulate specific dynein functions.     

Single-molecule analysis of dynein processivity and stepping behavior

Cytoplasmic dynein, the 1.2 MDa motor driving minus-end-directed motility, has been reported to move processively along microtubules, but its mechanism of motility remains poorly understood. Here, using S. cerevisiae to produce recombinant dynein with a chemically controlled dimerization switch, we show by structural and single-molecule analysis that processivity requires two dynein motor domains but not dynein's tail domain or any associated subunits. Dynein advances most frequently in 8 nm steps, although longer as well as side and backward steps are observed. Individual motor domains show a different stepping pattern, which is best explained by the two motor domains shuffling in an alternating manner between rear and forward positions. Our results suggest that cytoplasmic dynein moves processively through the coordination of its two motor domains, but its variable step size and direction suggest a considerable diffusional component to its step, which differs from Kinesin-1 and is more akin to myosin VI.     

Stable kinesin and dynein assemblies drive the axonal transport of mammalian prion protein vesicles

Kinesin and dynein are opposite-polarity microtubule motors that drive the tightly regulated transport of a variety of cargoes. Both motors can bind to cargo, but their overall composition on axonal vesicles and whether this composition directly modulates transport activity are unknown. Here we characterize the intracellular transport and steady-state motor subunit composition of mammalian prion protein (PrP(C)) vesicles. We identify Kinesin-1 and cytoplasmic dynein as major PrP(C) vesicle motor complexes and show that their activities are tightly coupled. Regulation of normal retrograde transport by Kinesin-1 is independent of dynein-vesicle attachment and requires the vesicle association of a complete Kinesin-1 heavy and light chain holoenzyme. Furthermore, motor subunits remain stably associated with stationary as well as with moving vesicles. Our data suggest a coordination model wherein PrP(C) vesicles maintain a stable population of associated motors whose activity is modulated by regulatory factors instead of by structural changes to motor-cargo associations.     

UNC-16, a JNK-signaling scaffold protein, regulates vesicle transport in C. elegans.

Transport of synaptic components is a regulated process. Loss-of-function mutations in the C. elegans unc-16 gene result in the mislocalization of synaptic vesicle and glutamate receptor markers. unc-16 encodes a homolog of mouse JSAP1/JIP3 and Drosophila Sunday Driver. Like JSAP1/JIP3, UNC-16 physically interacts with JNK and JNK kinases. Deletion mutations in Caenorhabditis elegans JNK and JNK kinases result in similar mislocalization of synaptic vesicle markers and enhance weak unc-16 mutant phenotypes. unc-116 kinesin heavy chain mutants also mislocalize synaptic vesicle markers, as well as a functional UNC-16::GFP. Intriguingly, unc-16 mutations partially suppress the vesicle retention defect in unc-104 KIF1A kinesin mutants. Our results suggest that UNC-16 may regulate the localization of vesicular cargo by integrating JNK signaling and kinesin-1 transport.     

p150Glued, the largest subunit of the dynactin complex, is nonessential in Neurospora but required for nuclear distribution.

Dynactin is a multisubunit complex that is required for cytoplasmic dynein, a minus-end-directed, microtubule-associated motor, to efficiently transport vesicles along microtubules in vitro. p150Glued, the largest subunit of dynactin, has been identified in vertebrates and Drosophila and recently has been shown to interact with cytoplasmic dynein intermediate chains in vitro. The mechanism by which dynactin facilitates cytoplasmic dynein-dependent vesicle transport is unknown. We have devised a genetic screen for cytoplasmic dynein/dynactin mutants in the filamentous fungus Neurospora crassa. In this paper, we report that one of these mutants, ro-3, defines a gene encoding an apparent homologue of p150Glued, and we provide genetic evidence that cytoplasmic dynein and dynactin interact in vivo. The major structural features of vertebrate and Drosophila p150Glued, a microtubule-binding site at the N-terminus and two large alpha-helical coiled-coil regions contained within the distal two-thirds of the polypeptide, are conserved in Ro3. Drosophila p150Glued is essential for viability; however, ro-3 null mutants are viable, indicating that dynactin is not an essential complex in N. crassa. We show that N. crassa cytoplasmic dynein and dynactin mutants have abnormal nuclear distribution but retain the ability to organize cytoplasmic microtubules and actin in anucleate hyphae.     

Dynactin is required for coordinated bidirectional motility, but not for dynein membrane attachment

Transport of cellular and neuronal vesicles, organelles, and other particles along microtubules requires the molecular motor protein dynein (Mallik and Gross, 2004). Critical to dynein function is dynactin, a multiprotein complex commonly thought to be required for dynein attachment to membrane compartments (Karki and Holzbaur, 1999). Recent work also has found that mutations in dynactin can cause the human motor neuron disease amyotrophic lateral sclerosis (Puls et al., 2003). Thus, it is essential to understand the in vivo function of dynactin. To test directly and rigorously the hypothesis that dynactin is required to attach dynein to membranes, we used both a Drosophila mutant and RNA interference to generate organisms and cells lacking the critical dynactin subunit, actin-related protein 1. Contrary to expectation, we found that apparently normal amounts of dynein associate with membrane compartments in the absence of a fully assembled dynactin complex. In addition, anterograde and retrograde organelle movement in dynactin deficient axons was completely disrupted, resulting in substantial changes in vesicle kinematic properties. Although effects on retrograde transport are predicted by the proposed function of dynactin as a regulator of dynein processivity, the additional effects we observed on anterograde transport also suggest potential roles for dynactin in mediating kinesin-driven transport and in coordinating the activity of opposing motors (King and Schroer, 2000).     

Microtubule binding by dynactin is required for microtubule organization but not cargo transport

Dynactin links cytoplasmic dynein and other motors to cargo and is involved in organizing radial microtubule arrays. The largest subunit of dynactin, p150(glued), binds the dynein intermediate chain and has an N-terminal microtubule-binding domain. To examine the role of microtubule binding by p150(glued), we replaced the wild-type p150(glued) in Drosophila melanogaster S2 cells with mutant DeltaN-p150 lacking residues 1-200, which is unable to bind microtubules. Cells treated with cytochalasin D were used for analysis of cargo movement along microtubules. Strikingly, although the movement of both membranous organelles and messenger ribonucleoprotein complexes by dynein and kinesin-1 requires dynactin, the substitution of full-length p150(glued) with DeltaN-p150(glued) has no effect on the rate, processivity, or step size of transport. However, truncation of the microtubule-binding domain of p150(glued) has a dramatic effect on cell division, resulting in the generation of multipolar spindles and free microtubule-organizing centers. Thus, dynactin binding to microtubules is required for organizing spindle microtubule arrays but not cargo motility in vivo.     

Disruption of axonal transport and neuronal viability by amyloid precursor protein mutations in Drosophila.

We tested the hypothesis that amyloid precursor protein (APP) and its relatives function as vesicular receptor proteins for kinesin-I. Deletion of the Drosophila APP-like gene (Appl) or overexpression of human APP695 or APPL constructs caused axonal transport phenotypes similar to kinesin and dynein mutants. Genetic reduction of kinesin-I expression enhanced while genetic reduction of dynein expression suppressed these phenotypes. Deletion of the C terminus of APP695 or APPL, including the kinesin binding region, disrupted axonal transport of APP695 and APPL and abolished the organelle accumulation phenotype. Neuronal apoptosis was induced only by overexpression of constructs containing both the C-terminal and Abeta regions of APP695. We discuss the possibility that axonal transport disruption may play a role in the neurodegenerative pathology of Alzheimer's disease.     

Regulating intraflagellar transport

Kinesin-2 motors mediate anterograde intraflagellar transport (IFT) of IFT particles from the ciliary base to its tip, where particles are remodelled before retrograde transport by dynein 2 motors. Bardet-Biedl syndrome (BBS) and IFT-A proteins are now implicated in regulation of IFT assembly at the ciliary base and tip.     

A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity

The timely delivery of membranous organelles and macromolecules to specific locations within the majority of eukaryotic cells depends on microtubule-based transport. Here we describe a screening method to identify mutations that have a critical effect on intracellular transport and its regulation using mutagenesis, multicolor-fluorescence microscopy, and multiplex genome sequencing. This screen exploits the filamentous fungus Aspergillus nidulans, which has many of the advantages of yeast molecular genetics but uses long-range microtubule-based transport in a manner more similar to metazoan cells. Using this method, we identified seven mutants that represent novel alleles of components of the intracellular transport machinery: specifically, kinesin-1, cytoplasmic dynein, and the dynein regulators Lis1 and dynactin. The two dynein mutations identified in our screen map to dynein''s AAA+ catalytic core. Single-molecule studies reveal that both mutations reduce dynein''s velocity in vitro. In vivo these mutants severely impair the distribution and velocity of endosomes, a known dynein cargo. In contrast, another dynein cargo, the nucleus, is positioned normally in these mutants. These results reveal that different dynein functions have distinct stringencies for motor performance.