Dynein mediates retrograde neurofilament transport within axons and anterograde delivery of NFs from perikarya into axons: regulation by multiple phosphorylation events

We examined the respective roles of dynein and kinesin in axonal transport of neurofilaments (NFs). Differentiated NB2a/d1 cells were transfected with green fluorescent protein-NF-M (GFP-M) and dynein function was inhibited by co-transfection with a construct expressing myc-tagged dynamitin, or by intracellular delivery of purified dynamitin and two antibodies against dynein's cargo domain. Monitoring of the bulk distribution of GFP signal within axonal neurites, recovery of GFP signal within photobleached regions, and real-time monitoring of individual NFs/punctate structures each revealed that pertubation of dynein function inhibited retrograde transport and accelerated anterograde, confirming that dynein mediated retrograde axonal transport, while intracellular delivery of two anti-kinesin antibodies selectively inhibited NF anterograde transport. In addition, dynamitin overexpression inhibited the initial translocation of newly-expressed NFs out of perikarya and into neurites, indicating that dynein participated in the initial anterograde delivery of NFs into neurites. Delivery of NFs to the axon hillock inner plasma membrane surface, and their subsequent translocation into neurites, was also prevented by vinblastine-mediated inhibition of microtubule assembly. These data collectively suggest that some NFs enter axons as cargo of microtubues that are themselves undergoing transport into axons via dynein-mediated interactions with the actin cortex and/or larger microtubules. C-terminal NF phosphorylation regulates motor association, since anti-dynein selectively coprecipitated extensively phosphorylated NFs, while anti-kinesin selectively coprecipitated less phosphorylated NFs. In addition, however, the MAP kinase inhibitor PD98059 also inhibited transport of a constitutively-phosphorylated NF construct, indicating that one or more additional, non-NF phosphorylation events also regulated NF association with dynein or kinesin.     

Microtubule motors,phosphorylation and axonal transport of neurofilaments

The recent demonstration that the axonal transport motors kinesin and dynein participate in axonal transport of neurofilaments (NFs), and that the association of NFs with these motors is regulated by phosphorylation provides new insight into several aspects of axonal transport and NF biology. This review juxtaposes older and more recent findings on NF dynamics, and speculates on the organization of axonal NFs as suggested by real-time analyses of NF transport.     

Regulation of neurofilament axonal transport by phosphorylation in optic axons in situ

Axonal transport of neurofilament (NFs) is considered to be regulated by phosphorylation. While existing evidence for this hypothesis is compelling, supportive studies have been largely restricted to correlative evidence and/or experimental systems involving mutants. We tested this hypothesis in retinal ganglion cells of normal mice in situ by comparing subunit transport with regional phosphorylation state coupled with inhibition of phosphatases. NF subunits were radiolabeled by intravitreal injection of 35S-methionine. NF axonal transport was monitored by following the location of the peak of radiolabeled subunits immunoprecipitated from 9x1.1 mm segments of optic axons. An abrupt decline transport rate was observed between days 1 and 6, which corresponded to translocation of the peak of radiolabeled subunits from axonal segment 2 into segment 3. Notably, this is far downstream from the only caliber increase of optic axons at 150 mu from the retina. Immunoblot analysis demonstrated a unique threefold increase between segments 2 and 3 in levels of a "late-appearing" C-terminal NF-H phospho-epitope (RT97). Intravitreal injection of the phosphatase inhibitor okadaic acid increased RT97 immunoreactivity within retinas and proximal axons, and markedly decreased NF transport rate out of retinas and proximal axons. These findings provide in situ experimental evidence for regulation of NF transport by site-specific phosphorylation.     

Abnormal neurofilament transport caused by targeted disruption of neuronal kinesin heavy chain KIF5A

To test the hypothesis that fast anterograde molecular motor proteins power the slow axonal transport of neurofilaments (NFs), we used homologous recombination to generate mice lacking the neuronal-specific conventional kinesin heavy chain, KIF5A. Because null KIF5A mutants die immediately after birth, a synapsin-promoted Cre-recombinase transgene was used to direct inactivation of KIF5A in neurons postnatally. Three fourths of such mutant mice exhibited seizures and death at around 3 wk of age; the remaining animals survived to 3 mo or longer. In young mutant animals, fast axonal transport appeared to be intact, but NF-H, as well as NF-M and NF-L, accumulated in the cell bodies of peripheral sensory neurons accompanied by a reduction in sensory axon caliber. Older animals also developed age-dependent sensory neuron degeneration, an accumulation of NF subunits in cell bodies and a reduction in axons, loss of large caliber axons, and hind limb paralysis. These data support the hypothesis that a conventional kinesin plays a role in the microtubule-dependent slow axonal transport of at least one cargo, the NF proteins.     

Growth cones contain a dynamic population of neurofilament subunits

Neurofilaments (NFs) are classically considered to transport in a primarily anterograde direction along axons, and to undergo bulk degradation within the synapse or growth cone (GC). We compared overall NF protein distribution with that of newly expressed NF subunits within NB2a/d1 cells by transfection with a construct encoding green fluorescent protein (GFP) conjugated NF-M subunits. GCs lacked phosphorylated NF epitopes, and steady-state levels of non-phosphosphorylated NF subunits within GC were markedly reduced compared to those of neurite shaft as indicated by conventional immunofluorescence. However, GCs contained significant levels of GFP-tagged subunits in the form of punctate or short filamentous structures that in some cases exceeded that visualized along the shaft itself, suggesting that GCs contained a relatively higher concentration of newly synthesized subunits. GFP-tagged NF subunits within GCs co-localized with non-phosphorylated NF immunoreactivity. GFP-tagged subunits were observed within GC filopodia in which steady-state levels of NF subunits were too low to be detected by conventional immunofluorescence. Selective localization of fluorescein versus rhodamine fluorescene was observed within GCs following expression of NF-M conjugated to DsRed1-E5, which shifts from fluorescein to rhodamine fluorescence within hours after expression; axonal shafts contained a more even distribution of fluorescein and rhodamine fluorescence, further indicating that GCs contained relatively higher levels of the most-recently expressed subunits. GFP-tagged structures were rapidly extracted from GCs under conditions that preserved axonal structures. These short filamentous and punctate structures underwent rapid bi-directional movement within GCs. Movement of GFP-tagged structures within GCs ceased following application of nocodazole, cytochalasin B, and the kinase inhibitor olomoucine, indicating that their motility was dependent upon microtubules and actin and, moreover, was due to active transport rather than simple diffusion. Treatment with the protease inhibitor calpeptin increased overall NF subunits, but increased those within the GC to a greater extent than those along the shaft, indicating that subunits in the GC undergo more rapid turnover than do those within the shaft. Some GCs contained coiled aggregates of GFP-tagged NFs that appeared to be contiguous with axonal NFs. NFs extended from these aggregates into the advancing GC as axonal neurites elongated. These data are consistent with the presence of a population of dynamic NF subunits within GCs that is apparently capable of participating in regional filament formation during axonal elongation, and support the notion that NF polymerization and transport need not necessarily occur in a uniform proximal-distal manner.     

Slow axonal transport mechanisms move neurofilaments relentlessly in mouse optic axons

Pulse-labeling studies of slow axonal transport in many kinds of axons (spinal motor, sensory ganglion, oculomotor, hypoglossal, and olfactory) have led to the inference that axonal transport mechanisms move neurofilaments (NFs) unidirectionally as a single continuous kinetic population with a diversity of individual transport rates. One study in mouse optic axons (Nixon, R. A., and K. B. Logvinenko. 1986. J. Cell Biol. 102:647-659) has given rise to the different suggestion that a significant and distinct population of NFs may be entirely stationary within axons. In mouse optic axons, there are relatively few NFs and the NF proteins are more lightly labeled than other slowly transported slow component b (SCb) proteins (which, however, move faster than the NFs); thus, in mouse optic axons, the radiolabel of some of these faster-moving SCb proteins may confuse NF protein analyses that use one dimensional (1-D) SDS-PAGE, which separates proteins by size only. To test this possibility, we used a 2-mm "window" (at 3-5 mm from the posterior of the eye) to compare NF kinetics obtained by 1-D SDS-PAGE and by the higher resolution two-dimensional (2-D) isoelectric focusing/SDS-PAGE, which separates proteins both by their net charge and by their size. We found that 1-D SDS-PAGE is insufficient for definitive NF kinetics in the mouse optic system. By contrast, 2-D SDS-PAGE provides essentially pure NF kinetics, and these indicate that in the NF-poor mouse optic axons, most NFs advance as they do in other, NF-rich axons. In mice, greater than 97% of the radiolabeled NFs were distributed in a unimodal wave that moved at a continuum of rates, between 3.0 and 0.3 mm/d, and less than 0.1% of the NF population traveled at the very slowest rates of less than 0.005 mm/d. These results are inconsistent with the proposal (Nixon and Logvinenko, 1986) that 32% of the transported NFs remain within optic axons in an entirely stationary state. As has been found in other axons, the axonal transport system of mouse optic axons moves NFs and other cytoskeletal elements relentlessly from the cell body to the axon tip.     

Antagonistic Roles of Neurofilament Subunits NF-H and NF-M Against NF-L in Shaping Dendritic Arborization in Spinal Motor Neurons

Dendrites play important roles in neuronal function. However, the cellular mechanism for the growth and maintenance of dendritic arborization is unclear. Neurofilaments (NFs), a major component of the neuronal cytoskeleton, are composed of three polypeptide subunits, NF-H, NF-M, and NF-L, and are abundant in large dendritic trees. By overexpressing each of the three NF subunits in transgenic mice, we altered subunit composition and found that increasing NF-H and/or NF-M inhibited dendritic arborization, whereas increasing NF-L alleviated this inhibition. Examination of cytoskeletal organization revealed that increasing NF-H and/or NF-M caused NF aggregation and dissociation of the NF network from the microtubule (MT) network. Increasing NF-H or NF-H together with NF-M further reduced NFs from dendrites. However, these changes were reversed by elevating the level of NF-L with either NF-H or NF-M. Thus, NF-L antagonizes NF-H and NF-M in organizing the NF network and maintaining a lower ratio of NF-H and NF-M to NF-L is critical for the growth of complex dendritic trees in motor neurons.     

Hypophosphorylated neurofilament subunits undergo axonal transport more rapidly than more extensively phosphorylated subunits in situ

Axonal transport of neurofilaments (NFs) has long been considered to be regulated by phosphorylation. We present evidence that in optic axons of normal mice, the rate of NF axonal transport is inversely correlated with the NF phosphorylation state. In addition to 200 kDa NF-H and 145 kDa NF-M, axonal cytoskeletons from CNS contained a range of phospho-variants of NF-H migrating between 160-200 kDa, and of NF-M migrating at 97-145 kDa. While 160 kDa phospho-variants of NF-H have been well characterized, we confirmed the identity of the previously-described 97 kDa species as a hypophospho-variant of NF-M since (1) pulse-chase metabolic labeling confirmed the 97 kDa species to be a new synthesis product that was converted by phosphorylation over time into a form migrating at 145 kDa, (2) the 97 kDa protein reacted with multiple NF-M antibodies, including one specific for hypophosphorylated NF-M, and (3) dephosphorylation converted NF-M isoforms to 97 kDa. Autoradiographic analyses following metabolic radiolabeling demonstrated that hypophosphorylated NF-H and NF-M isoforms underwent substantially more rapid transport in situ than did extensively phosphorylated isoforms, while NF-H subunits bearing a developmentally delayed C-terminal phospho-epitope transported at a rate slower than that of total 200 kDa NF-H. Differential transport of phospho-variants also highlights that these variants are not homogeneously distributed among NFs, but are segregated to some extent among distinct, although probably overlapping, NF populations, indicating that axonal NFs are not homogeneous with respect to phosphorylation state.     

Selective accumulation of the high molecular weight neurofilament subunit within the distal region of growing axonal neurites.

Axonal maturation in situ is accompanied by the transition of neurofilaments (NFs) comprised of only NF-M and NF-L to those also containing NF-H. Since NF-H participates in interactions of NFs with each other and with other cytoskeletal constituents, its appearance represents a critical event in the stabilization of axons that accompanies their maturation. Whether this transition is effected by replacement of "doublet" NFs with "triplet" NFs, or by incorporation of NF-H into existing doublet NFs is unclear. To address this issue, we examined the distribution of NF subunit immunoreactivity within axonal cytoskeletons of differentiated NB2a/d1 cell and DRG neurons between days 3-7 of outgrowth. Endogenous immunoreactivity either declined in a proximal-distal gradient or was relatively uniform along axons. This distribution was paralleled by microinjected biotinylated NF-L. By contrast, biotinylated NF-H displayed a bipolar distribution, with immunoreactivity concentrated within the proximal- and distal-most axonal regions. Proximal biotinylated NF-H accumulation paralleled that of endogenous NF immunoreactivity; however, distal-most biotinylated NF-H accumulation dramatically exceeded that of endogenous NFs and microinjected NF-L. This phenomenon was not due to co-polymerization of biotin-H with vimentin or alpha-internexin. This phenomenon declined with continued time in culture. These data suggest that NF-H can incorporate into existing cytoskeletal structures, and therefore suggest that this mechanism accounts for at least a portion of the accumulation of triplet NFs during axonal maturation. Selective NF-H accumulation into existing cytoskeletal structures within the distal-most region may provide de novo cytoskeletal stability for continued axon extension and/or stabilization.     

The single neurofilament subunit of the lamprey forms filaments and regulates axonal caliber and neuronal size in vivo

Neurofilaments (NFs) are composed of a heteropolymer of three related subunits in mammalian neurons, where they are a major component of the cytoskeleton in large neurons and are thought to regulate axonal diameter. NFs in the lamprey, while ultrastructurally and functionally indistinguishable from mammalian NFs, are polymers of a single subunit protein, NF180. In this study, we use the simplicity of lamprey NFs and the accessibility of the lamprey central nervous system (CNS) to examine the effects of overproducing NFs in an identified giant neuron in vivo, and thus to elucidate the role of NFs in regulating neuronal size and axonal caliber in the vertebrate CNS. We show that overexpression of NF180 tagged with a variant of Green Fluorescent Protein (EYFP) in identified lamprey neurons (ABCs) and in human neuroblastoma (NB2a) cells results in the assembly of exogenous NF180 into ultrastructurally normal NFs that are tightly packed and unphosphorylated. These accumulate in the somata of NB2a cells and produce somatic swelling by 3 days post-transfection. NF180 overexpression in lamprey ABCs in vivo causes exogenous NFs to accumulate in ABC axons, somata, and dendrites, and induces a significant increase in axonal diameter without increasing axonal NF packing density. Overexpression of EYFP alone has none of these effects. We conclude that NF180 normally plays a critical role in determining axonal caliber in ABCs and may influence neuronal size in situations where NFs accumulate in the soma, such as after axonal injury.     

Tau inhibits anterograde axonal transport and perturbs stability in growing axonal neurites in part by displacing kinesin cargo: neurofilaments attenuate tau-mediated neurite instability

Overexpression of tau compromises axonal transport and induces retraction of growing neurites. We tested the hypothesis that increased stability provided by neurofilaments (NFs) may prevent axonal retraction. NB2a/d1 cells were differentiated for 3 days, at which time phosphorylated NFs appear and for 14 days, which induces continued neurite elongation and further phospho-NF accumulation. Cultures were transfected with a construct that expresses full-length, 4-repeat tau. Consistent with prior studies, overexpression of tau induced retraction of day three axonal neurites even following treatment with the microtubule-stabilizing drug taxol. Axonal neurites of day 14 cells were more resistant to tau-mediated retraction. To test whether or not this resistance was derived from their additional NF content, day 3 cultures were co-transfected with constructs expressing tau and NF-M (which increases overall axonal NFs). Overexpression of NF-M attenuated tau-mediated retraction of day 3 axonal neurites. By contrast, co-transfection with constructs expressing tau and vimentin (which increases axonal neurites length) did not attenuate tau-mediated neurite retraction. Co-precipitation experiments indicate that tau is a cargo of kinesin, and that tau overexpression may displace other kinesin-based cargo, including both critical cytoskeletal proteins and organelles. However, cultures simultaneously transfected with constructs expressing NF-M and tau, the level of examined vesicles was maintained. These collectively indicate that NFs stabilize developing axonal neurites and can counteract the destabilizing force resulting from overexpression of tau, and underscore that the development and stabilization of axonal neurites is dependent upon a balance of cytoskeletal elements.     

Production of Monoclonal Antibodies Against Neurofilament-Associated Proteins: Demonstration of Association with Neurofilaments by a Coimmunoprecipitation Method

Abstract: A panel of monoclonal antibodies (MAbs) was produced against mouse brain proteins that bind to the tail domain of the neurofilament (NF) heavy (200-kDa) subunit (NF-H) in vitro. An in vivo association of the MAb ligands with NFs was confirmed by examining reactivity of the MAbs with immunoprecipitated NF-H complexes. Using this method we demonstrated association of the ligands of three of the MAbs with NFs. In contrast, glial fibrillary acidic protein and an unknown 97-kDa brain protein were not associated with NFs by this criterion. An 80-kDa doublet that coimmunoprecipitated with NF-H complexes, recognized by MAb 223, was shown by immunocytochemistry and immunoblotting to be synapsin Ia and Ib. Using a complementary approach, we confirmed an association of synapsin with NFs by demonstrating that immunoprecipitated synapsin I complexes contained NF-H and NF medium (160-kDa) subunits. MAbs 63 and 105 recognized a more complex set of proteins that had predominantly synaptic localizations. These data suggest that NFs may provide important support for attachment and/or transport of synaptic proteins in brain.     

Axonal neurofilaments differ in composition and morphology from those in the soma of the squid stellate ganglion

Triton X-100 insoluble neurofilament (NF) fractions were obtained from two parts of the stellate ganglion and the main giant axon. These were analyzed by one- and two-dimensional gradient polyacrylamide gel electrophoresis, cyclic assembly and disassembly, and electron microscopy. The NF fractions from the ganglion cell bodies (GCB) and from the part of the ganglion mainly consisting of axon initial segments (GIS) were of similar composition; neither contained detectable amounts of the 220 kda and high molecular weight (greater than 400 kda) NF subunits that were prominent in the axonal NF fraction. However, the GCB and GIS did contain large quantities of a set of 65 kda polypeptides that were minor constituents of the axonal NF fraction. The 65 kda-containing NF fraction from the ganglion could be cyclically disassembled and reassembled, but only under low salt conditions, in contrast to the high salt conditions used to cycle axonal NFs. A comparison of the peptide map of the 65 kda polypeptides with that of the 60 kda axonal NF subunit showed them to be different. These biochemical differences between the ganglionic and axonal NF fractions correlated with morphologic distinctions: ganglionic NFs were relatively smooth surfaced, whereas axonal NFs had long sidearms. Such observations support the hypothesis that the NF cytoskeleton of the neuron soma is different from that of the axon. Furthermore, the change from the somal form to the axonal form of NFs appears to occur in the region where the axon initial segment increases in diameter to become the axon proper.     

The predominant form in which neurofilament subunits undergo axonal transport varies during axonal initiation, elongation, and maturation

The forms in which neurofilament (NF) subunits undergo axonal transport is controversial. Recent studies from have provided real-time visualization of the slow axonal transport of NF subunits by transfecting neuronal cultures with constructs encoding green fluorescent protein (GFP)-conjugated NF-M subunits. In our studies in differentiated NB2a/d1 cells, the majority NF subunits underwent transport in the form of punctate NF precursors, while studies in cultured neurons have demonstrated transport of NF subunits in predominantly filamentous form. Although different constructs were used in these studies, transfection of the same cultured neurons with our construct yielded the filamentous pattern observed by others, while transfection of our cultures with their construct generated punctate structures, confirming that the observed differences did not reflect variances in assembly-competence among the constructs. Manipulation of intracellular kinase, phosphatase, and protease activities shifted the predominant form of GFP-conjugated subunits between punctate and filamentous, confirming, as shown previously for vimentin, that punctate structures represent precursors for intermediate filament formation. Since these prior studies were conducted at markedly differing neuronal differentiation states, we tested the alternate hypothesis that these differing results reflected developmental alterations in NF dynamics that accompany various stages of neuritogenesis. We conducted time-course analyses of transfected NB2a/d1 cells, including monitoring of transfected cells over several days, as well as transfecting cells at varying intervals prior to and following induction of differentiation and axonal neurite outgrowth. GFP-conjugated subunits were predominantly filamentous during the period of most robust axonal outgrowth and NF accumulation, and presented a mixed profile of punctate and filamentous forms prior to neuritogenesis and following the developmental slowing of neurite outgrowth. These analyses demonstrate that NF subunits are capable of undergoing axonal transport in multiple forms, and that the predominant form in which NF subunits undergo axonal transport varies in accord with the rate of axonal elongation and accumulation of NFs within developing axons.     

Regulation of the transition from vimentin to neurofilaments during neuronal differentiation

Vimentin (Vm) is initially expressed by nearly all neuronal precursors in vivo, and is replaced by neurofilaments (NFs) shortly after the immature neurons become post-mitotic. Both Vm and NFs can be transiently detected within the same neurite, and Vm is essential for neuritogenesis at least in culture. How neurons effect the orderly transition from expression of Vm as their predominant intermediate filament to NFs remains unclear. We examined this phenomenon within growing axonal neurites of NB2a/d1 cells. Transfection of cells with a construct expressing Vm conjugated to green fluorescent protein confirmed that axonal transport machinery for Vm persisted following the developmental decrease in Vm, but that the amount undergoing transport decreased in parallel to the observed developmental increase in NF transport. Immunoprecipitation from pulse-chase radiolabeled cells demonstrated transient co-precipitation of newly synthesized NF-H with Vm, followed by increasing co-precipitation with NF-L. Immunofluorescent and immuno-electron microscopic analyses demonstrated that some NF and Vm subunits were incorporated into the same filamentous profiles, but that Vm was excluded from the longitudinally-oriented "bundle" of closely-apposed NFs that accumulates within developing axons and is known to undergo slower turnover than individual NFs. These data collectively suggest that developing neurons are able to replace their Vm-rich cytoskeleton with one rich in NFs simply by down-regulation of Vm expression and upregulation of NFs, coupled with turnover of existing Vm filaments and Vm-NF heteropolymers.     

Axonal transport of amyloid precursor protein is mediated by direct binding to the kinesin light chain subunit of kinesin-I

We analyzed the mechanism of axonal transport of the amyloid precursor protein (APP), which plays a major role in the development of Alzheimer's disease. Coimmunoprecipitation, sucrose gradient, and direct in vitro binding demonstrated that APP forms a complex with the microtubule motor, conventional kinesin (kinesin-I), by binding directly to the TPR domain of the kinesin light chain (KLC) subunit. The estimated apparent Kd for binding is 15-20 nM, with a binding stoichiometry of two APP per KLC. In addition, association of APP with microtubules and axonal transport of APP is greatly decreased in a gene-targeted mouse mutant of the neuronally enriched KLC1 gene. We propose that one of the normal functions of APP may be as a membrane cargo receptor for kinesin-I and that KLC is important for kinesin-I-driven transport of APP into axons.     

Endogenous GSK‐3/Shaggy Regulates Bidirectional Axonal Transport of the Amyloid Precursor Protein

Neurons rely on microtubule (MT) motor proteins such as kinesin‐1 and dynein to transport essential cargos between the cell body and axon terminus. Defective axonal transport causes abnormal axonal cargo accumulations and is connected to neurodegenerative diseases, including Alzheimer's disease (AD). Glycogen synthase kinase 3 (GSK‐3) has been proposed to be a central player in AD and to regulate axonal transport by the MT motor protein kinesin‐1. Using genetic, biochemical and biophysical approaches in Drosophila melanogaster, we find that endogenous GSK‐3 is a required negative regulator of both kinesin‐1‐mediated and dynein‐mediated axonal transport of the amyloid precursor protein (APP), a key contributor to AD pathology. GSK‐3 also regulates transport of an unrelated cargo, embryonic lipid droplets. By measuring the forces motors generate in vivo, we find that GSK‐3 regulates transport by altering the activity of kinesin‐1 motors but not their binding to the cargo. These findings reveal a new relationship between GSK‐3 and APP, and demonstrate that endogenous GSK‐3 is an essential in vivo regulator of bidirectional APP transport in axons and lipid droplets in embryos. Furthermore, they point to a new regulatory mechanism in which GSK‐3 controls the number of active motors that are moving a cargo .     

Role of cytoplasmic dynein in the axonal transport of microtubules and neurofilaments

Recent studies have shown that the transport of microtubules (MTs) and neurofilaments (NFs) within the axon is rapid, infrequent, asynchronous, and bidirectional. Here, we used RNA interference to investigate the role of cytoplasmic dynein in powering these transport events. To reveal transport of MTs and NFs, we expressed EGFP-tagged tubulin or NF proteins in cultured rat sympathetic neurons and performed live-cell imaging of the fluorescent cytoskeletal elements in photobleached regions of the axon. The occurrence of anterograde MT and retrograde NF movements was significantly diminished in neurons that had been depleted of dynein heavy chain, whereas the occurrence of retrograde MT and anterograde NF movements was unaffected. These results support a cargo model for NF transport and a sliding filament model for MT transport.     

Follicle separation during Drosophila oogenesis requires the activity of the kinesin II-associated polypeptide Kap in germline cells

Cellular localization of organelles, protein complexes and single mRNAs depends on the directed transport along microtubule tracks, a process mediated by ATP-driven molecular motor proteins of the dynein and kinesin superfamilies. Kinesin II is a heterotrimeric protein complex composed of two motor subunits and a unique nonmotor Kinesin-associated protein (Kap). Kap was shown to transport both particulate cargo, as axoneme components in rafts, and membrane-bounded organelles such as melanosomes. Drosophila Kinesin II was shown to be essential for the axonal transport of choline acetyltransferase in a specific set of neurons. We have generated Kap mutants and show that gene activity is not only required for neuronal function but also for separation of follicles during early oogenesis. The data suggest that Kap participates in the transport of signalling components required for instructive interactions between germline and soma cells.