InteractiveFly: GeneBrief

Kinesin-like protein at 64D & Kinesin-like protein at 68D: Biological Overview | References

Gene names - Kinesin-like protein at 64D & Kinesin-like protein at 68D

Synonyms - Kinesin-2 and Kinesin2B for Klp64D and Kinesin-2 for Klp68D

Cytological map positions - 64C13-64C13 & 68D6-68E1

Function - cytoskeletal motor protein

Keywords - Klp64D and Klp68D are kinesin-2α kinesin-2β motor subunits of the heterotrimeric kinesin-2. Klp64D associates with Klp68D and Kap3. Klp64D and Klp68D play an essential role in sensory cilia assembly and axonal transport, microtubule organization in dendrites, hearing

Symbols - Klp64D & Klp68D

FlyBase IDs: FBgn0004380 & FBgn0004381

Genetic map positions - chr3L:5,356,864-5,359,392 & chr3L:11,795,614-11,798,875

Classifications - Kinesin motor domain, kinesins II or KIF3_like proteins

Cellular location - cytoplasmic

NCBI links for Klp64D: EntrezGene | Nucleotide | Protein
NCBI links for Klp68D: EntrezGene | Nucleotide | Protein

Klp64D orthologs: Biolitmine
Klp68D orthologs: Biolitmine
Recent literature
Vuong, L. T., Iomini, C., Balmer, S., Esposito, D., Aaronson, S. A. and Mlodzik, M. (2018). Kinesin-2 and IFT-A act as a complex promoting nuclear localization of beta-catenin during Wnt signalling. Nat Commun 9(1): 5304. PubMed ID: 30546012
Wnt/Wg-signalling is critical signalling in all metazoans. Recent studies suggest that IFT-A proteins and Kinesin-2 modulate canonical Wnt/Wg-signalling independently of their ciliary role. Whether they function together in Wnt-signalling and their mechanistic role in the pathway remained unresolved. This study demonstrated that Kinesin-2 and IFT-A proteins act as a complex during Drosophila Wg-signalling, affecting pathway activity in the same manner, interacting genetically and physically, and co-localizing with beta-catenin, the mediator of Wnt/Wg-signalling on microtubules. Following pathway activation, Kinesin-2/IFT-A mutant cells exhibit high cytoplasmic beta-catenin levels, yet fail to activate Wg-targets. In mutant tissues in both, Drosophila and mouse/MEFs, nuclear localization of beta-catenin is markedly reduced. A conserved, motor-domain dependent function of the Kinesin-2/IFT-A complex is demonstrated in promoting nuclear translocation of beta-catenin. This is mediated by protecting beta-catenin from a conserved cytoplasmic retention process, thus identifying a mechanism for Kinesin-2/IFT-A in Wnt-signalling that is independent of their ciliary role.
Ahmed, Z., Doodhi, H., Bhaumik, A., Mazumdar, S. and Ray, K. (2019). The structural dynamics of the kinesin-2 stalk heterodimer and its biological relevance. Biochem Biophys Res Commun 518(1): 171-177. PubMed ID: 31420166
Association between two motor subunits through the rod/stalk domain enables molecular motors to walk processively on protein filaments. Previous studies suggested that structural flexibility in the coiled-coil stalk of kinesins is essential for processive runs. The stalk of heterotrimeric kinesin-2, a comparatively less processive motor, is unstable at ambient temperature. How this structural instability impacts the motor function is unclear. This study used the Forster Resonance Energy Transfer based assays to show that the Drosophila kinesin-2alpha/beta stalk heterodimer is dynamic at physiological conditions. Insertion of a missense mutation (Glu551-Lys) at the C-terminal half of kinesin-2alpha stalk reduces the dynamics of the heterodimeric stalk in vitro. The mutation, isolated as a recessive lethal allele in a forward genetic screen, is reported to disrupt the motor function in axonal transport and cilia development. Together these two results suggest that the dynamic instability of the kinesin-2 stalk could play a crucial role in maintaining its biological function.
Ahmed, Z., Mazumdar, S. and Ray, K. (2019). Kinesin associated protein, DmKAP, binding harnesses the C-terminal ends of the Drosophila kinesin-2 stalk heterodimer. Biochem Biophys Res Commun. PubMed ID: 31784087
The heterotrimeric kinesin-2 consists of two distinct motor subunits and an accessory protein, KAP, which binds to the coiled-coil stalk domains and one of the tail domains of the motor subunits. Genetic studies revealed that KAP is essential for the kinesin-2 functions in cilia, flagella, and axon. However, the structural significance of the KAP binding on kinesin-2 assembly and stability is not known. Uusing the Fluorescence Lifetime assay this study showed that DmKAP binding selectively reduces the distance between the C-terminal ends of Drosophila kinesin-2 stalk heterodimer. Insertion of a missense mutation (E551K) in the Drosophila kinesin-2alpha stalk fragment, which was shown to reduce the structural dynamics of the stalk heterodimer earlier, also reduced the distances at both the N- and C-terminal ends of the stalk heterodimer independent of DmKAP. The zipping effect, particularly at the N-terminal end of the mutant stalk heterodimer, is further enhanced in the presence of DmKAP. Together, these results suggest that the KAP binding could alter the structural dynamics of kinesin-2 stalk heterodimer at the C-terminal end and stabilize the association between the stalk domains.
Mukherjee, A., Brooks, P. S., Bernard, F., Guichet, A. and Conduit, P. T. (2020). Microtubules originate asymmetrically at the somatic golgi and are guided via Kinesin2 to maintain polarity within neurons. Elife 9. PubMed ID: 32657758
Neurons contain polarised microtubule arrays essential for neuronal function. How microtubule nucleation and polarity are regulated within neurons remains unclear. This study shows that γ-tubulin localises asymmetrically to the somatic Golgi within Drosophila neurons. Microtubules originate from the Golgi with an initial growth preference towards the axon. Their growing plus ends also turn towards and into the axon, adding to the plus-end-out microtubule pool. Any plus ends that reach a dendrite, however, do not readily enter, maintaining minus-end-out polarity. Both turning towards the axon and exclusion from dendrites depend on Kinesin-2, a plus-end-associated motor that guides growing plus ends along adjacent microtubules. It is proposed that Kinesin-2 engages with a polarised microtubule network within the soma to guide growing microtubules towards the axon; while at dendrite entry sites engagement with microtubules of opposite polarity generates a backward stalling force that prevents entry into dendrites and thus maintains minus-end-out polarity within proximal dendrites.
Vuong, L. T., Won, J. H., Nguyen, M. B. and Choi, K. W. (2020). Role of Armadillo repeat 2 and kinesin-II motor subunit Klp64D for wingless signaling in Drosophila. Sci Rep 10(1): 13864. PubMed ID: 32807823
Armadillo (Arm) is crucial for transducing Wingless (Wg) signaling. Previous work has shown that Klp64D, a motor subunit of Drosophila kinesin-II, interacts with Arm for Wg signaling. Molecular basis for this interaction has remained unknown. This study has identified a critical Arm repeat (AR) required for binding Klp64D and Wg signaling. Arm/β-catenin family proteins contain a conserved domain of 12 Arm repeats (ARs). Five of these ARs can interact with Klp64D, but only the second AR (AR2) binds to the cargo/tail domain of Klp64D. Overexpression of AR2 in wing imaginal disc is sufficient to cause notched wing margin. This phenotype by AR2 is enhanced or suppressed by reducing or increasing Klp64D expression, respectively. AR2 overexpression inhibits Wg signaling activity in TopFlash assay, consistent with its dominant-negative effects on Klp64D-dependent Wg signaling. Overexpression of the Klp64D cargo domain also results in dominant-negative wing notching. Genetic rescue data indicate that both AR2 and Klp64D cargo regions are required for the function of Arm and Klp64D, respectively. AR2 overexpression leads to an accumulation of Arm with GM130 Golgi marker in Klp64D knockdown. This study suggests that Wg signaling for wing development is regulated by specific interaction between AR2 and the cargo domain of Klp64D.

Cholinergic activity is essential for cognitive functions and neuronal homeostasis. Choline Acetyltransferase (ChAT), a soluble protein that synthesizes acetylcholine at the presynaptic compartment, is transported in bulk in the axons by the heterotrimeric Kinesin-2 motor. Axonal transport of soluble proteins is described as a constitutive process assisted by occasional, non-specific interactions with moving vesicles and motor proteins. This study reports that an increase in the influx of Kinesin-2 motor and association between ChAT and the motor during a specific developmental period enhances the axonal entry, as well as the anterograde flow of the protein, in the sensory neurons of intact Drosophila nervous system. Loss of cholinergic activity due to Hemicholinium and Bungarotoxin treatments, respectively, disrupts the interaction between ChAT and Kinesin-2 in the axon, and the episodic enhancement of axonal influx of the protein. Altogether, these observations highlight a phenomenon of synaptic activity-dependent, feedback regulation of a soluble protein transport in vivo, which could potentially define the quantum of its pre-synaptic influx (Dey, 2018).

Synaptic activity is essential for the development and maintenance of neuronal circuits. It regulates the presynaptic influx of vesicles and organelles. Several soluble proteins are selectively enriched in the axon and synapses. Transport of these proteins plays a major role in both the assembly and maintenance of synaptic activity. Also, the onset of several neuropathies is correlated to an abnormal transport of soluble proteins. However, little is known about the regulation of their transport in the axon. Currently, all soluble axonal transport phenomena are described as constitutive processes driven by either stochastic or non-specific interactions with motors or vesicular cargoes in the neighborhood. In contrast, soluble forms of Dynein and ChAT are transported directly by Kinesin-1 and Kinesin-2, respectively, towards the synapse. (Twelvetrees, 2016; Ligon, 2004; Sadananda, 2012). Although Dynein flux is constitutive, ChAT movement in axon acquires an anterograde bias contributed by the heterotrimeric Kinesin-2 during a certain developmental stage (Sadananda, 2012), resulting in the pre-synaptic enrichment of the protein in the central nervous system of Drosophila (Baqri, 2006). The existing slow transport hypotheses, however, cannot explain the episodic movement and regulated pre-synaptic influx of soluble ChAT (Dey, 2018).

Acetylcholine (ACh) mediated neurotransmission is implicated in several cognitive functions, and loss of cholinergic activity is indicated to cause dementia and neurodegenerative disorders. ACh is regenerated through the acetylation of choline by Choline acetyltransferase (ChAT), a soluble enzyme synthesized in the cell body and enriched at the presynaptic compartments. Local recruitment of cholinergic machinery was found to promote neurite outgrowth and maintenance of motor activity in zebrafish larvae. In Drosophila, a complete loss of zygotic ChAT function in the homozygous cha mutants caused progressive paralysis and lethality at nonpermissive temperatures (Greenspan, 1980; Yasuyama 1996), and an increased presynaptic localization of ChAT is suggested to promote synapse assembly in the ventral ganglia of larval brain (Dey, 2017). Interestingly, it also induced behavioral changes of Drosophila larvae, indicating a possible correlation between the altered transport and synaptic functioning (Dey, 2018).

Therefore, to understand the mechanism providing the anterograde bias to the bulk of ChAT movement and the impact of its presynaptic activity on the transport, interactions between ChAT and Kinesin-2 motor subunit were estimated in situ at different developmental stages. The cholinergic activity was perturbed using pharmacological reagents, and the effect on this transport was studied. A high-sensitivity detector was used for data acquisition that enhanced the signal-to-noise ratio substantially as compared to the earlier report (Sadananda, 2012). The results indicate that a temporally-restricted association with Kinesin-2, during 77-78 hours after egg laying (AEL), throughout the neuron drives the episodic movement of the bulk of ChAT towards synapse. A step increase in the axonal levels of the motor during 77-78 h AEL and cholinergic activity enhanced the entry and anterograde flux of ChAT in axons. The bulk movement of ChAT appears to evolve from a restricted to a directed, facilitated diffusion during this period (Dey, 2018).

Axonal transport of ChAT has been extensively studied in various organisms and neuron types. Estimates of accumulated ChAT activity at the ligature of rat sciatic nerve suggested that the enzyme flows anterogradely at an average rate of ~1.2 mm/day. Using the high-resolution FRAP this study estimated a much faster max flow rate (1.8 μm/s or ~155 mm/day) in the axons of intact lch5 neurons of Drosophila larvae, as compared to an earlier estimate (0.97 μm/s or ~83 mm/day) obtained from the short interneurons of the ventral ganglion. A similar disparity in rates was reported for the other slow axonal cargoes such as neurofilaments, CaMKII, Synapsin, and Actin. This apparent discrepancy in the rate estimates is a consequence of spatiotemporal characteristics of the transport which are reflected in the assaying paradigms and acquisition parameters. Besides the 76-79 h AEL interval in the third instar stage, another episode of ChAT influx was observed in lch5 axons during 52-56 h AEL in the second instar stage. Assuming that ChAT transport episode is restricted to an hourly interval during each molt, the effective flow rate during a 24 h molting period would be 3.5 mm/day which correlates well to transport characteristics of ChAT as a slow rate component. These results are obtained from fully ensheathed functional neurons connected to the native circuitry at different developmental stages. Thus, it also provided near endogenous characteristics of the transport. With the improved observation capability, this study found that the temporal parameters of the ChAT transport are consistent in both the small interneurons of ventral ganglion, as well as in the mature lch5 neurons, suggesting that the episodic nature of the ChAT transport is an intrinsic property (Dey, 2018).

Nuclear Choline Acetyltransferase activates transcription of a high-affinity Choline transporter

ChAT was reported to bind directly to the C-terminal tail domain of the Kinesin-2α subunit in vitro (Dey, 2012). Kinesin-2 is essential for two distinct aspects of the transport process -- entry into the axons and for conferring the anterograde bias observed at 78 h AEL. The FRAP and FRET assay further suggested that the episodic movement of the bulk of ChAT is initiated through a transient association with the Kinesin-2 motor throughout the neuron. Although the motor was present in the axon all throughout, the association was limited to an hourly interval or less during late larval development. Studies showed that temporal switching of association from Kinesin-3 (Unc-104) to Dynein shifts the transport of Rab3 vesicles from anterograde to retrograde in the DD neurons of C. elegans. This change in the modality of Rab3 vesicle transport in the DD neurons was associated with synapse restructuring induced by cyclin, CYY-1 and the cyclin-dependent kinase, CDK5. The ChAT transport episodes appear to closely follow the molting cycle, which is induced through the surge of Juvenile Hormone (20-Hydroxyecdysterone) in Drosophila larvae. Considering the timescale of the transport modulation, certain post-translational modifications such as phosphorylation could enhance the affinity between ChAT and Kinesin-2. For example, Calmodulin Kinase II (CaMKII)-mediated phosphorylation of Kinesin-2 tail is suggested to increased the transport of N-Cadhern (Matsuo, 2011)

Anterograde transport of Rab4-associated vesicles regulates synapse organization in Drosophila.

Local endosomal recycling at synapses is essential to maintain neurotransmission. Rab4 GTPase, found on sorting endosomes, is proposed to balance the flow of vesicles among endocytic, recycling, and degradative pathways in the presynaptic compartment. This study reports that Rab4-associated vesicles move bidirectionally in Drosophila axons but with an anterograde bias, resulting in their moderate enrichment at the synaptic region of the larval ventral ganglion. Results from FK506 binding protein (FKBP) and FKBP-Rapamycin binding domain (FRB) conjugation assays in rat embryonic fibroblasts together with genetic analyses in Drosophila indicate that an association with Kinesin-2 (mediated by the tail domain of Kinesin-2α/KIF3A/KLP64D subunit) moves Rab4-associated vesicles toward the synapse. Reduction in the anterograde traffic of Rab4 causes an expansion of the volume of the synapse-bearing region in the ventral ganglion and increases the motility of Drosophila larvae. These results suggest that Rab4-dependent vesicular traffic toward the synapse plays a vital role in maintaining synaptic balance in this neuronal network (Dey, 2017).

The apical compartment of a neuronal cell extends into specific processes forming axons and synapses that are maintained by a distributed endosomal system. For example, vesicle recycling at the synapse, orchestrated by various RabGTPases and associated proteins, renews the readily releasable pool of synaptic vesicles sustaining the neurotransmission. Rab4 is thought to play an important role in balancing the vesicle traffic between the recycling and degradation pathways involved in various cellular processes such as metabolism, cell secretion, and antigen processing. Recruitment of Rab4 on sorting intermediate endosomes allows the transfer of vesicular cargoes from early endosomes to recycling endosomes in axons and at synapses. The activity of Rab4 has also been implicated in the progression of growth cone in Xenopus and the maintenance of dendritic spines in rat hippocampal neurons. Furthermore, endosomal abnormalities found in the cholinergic basal forebrain of patients with Alzheimer's disease correlate with elevated levels of Rab4. All these reports suggest that Rab4-dependent vesicle sorting is essential for the development and maintenance of the nervous system (Dey, 2017).

Vesicular cargoes, including the RabGTPases, originate at the trans-Golgi network, and microtubule-dependent motors transport them to different destinations within a cell. Specific association with motors ensures differential and dynamic subcellular localizations of various RabGTPases according to tissue-specific activities and metabolic demands. For instance, the anterograde trafficking of early, sorting, and late endosomes is dependent on the Kinesin-3 motors KIF13, KIF16B, and KIF1A/IBβ, respectively. Similarly, in Drosophila, Rab5-positive early endosomes associate with Khc-73, which is the ortholog of mammalian KIF13, showing a conserved machinery of Kinesin-Rab interaction. The same vesicles also associate with Dynein for their retrograde movement. It is further indicated that multiple RabGTPases can be grouped together and transported to a destination within a cell where they can engage in different functions. Although the long-range transport of certain presynaptic RabGTPases has been studied in cultured neurons, it is still unclear how the bidirectional movement of Rab-associated vesicles could localize them in distant compartments such as the synapse (Dey, 2017).

This study has shown that Rab4-associated vesicles are transported with an anterograde bias, induced by a specific interaction with the tail domain of Kinesin-2α subunit, which results in its moderate enrichment at the synapses. The rate of pre-synaptic inflow of Rab4 is positively correlated with its activation. Interestingly, this study found that a decreased flow of the Rab4-associated vesicles expanded the region occupied by synapses in the neuropil region of the larval ventral ganglion and enhanced larval motility. Altogether, these results indicate that the flow of Rab4-associated vesicles could maintain synaptic homeostasis in a neuronal network (Dey, 2017).

Heterotrimeric Kinesin-2, implicated in the trafficking of apical endosomes and late endosomal vesicles, was initially thought to bind to its cargoes through the accessory subunit KAP3. However, recent studies have reported that motor subunits could independently interact with soluble proteins through the tail domain. This study shows that the Kinesin-2α tail can also bind to a membrane-associated protein, Rab4. A preliminary investigation in the lab suggests that the interaction in not direct. The RabGTPases are known to recruit a variety of different motors to the membrane, directing intracellular trafficking. Although a previous report indicated that Rab4 could bind to Kinesin-2, this study found that such an interaction leads to the anterograde trafficking of Rab4-associated vesicles in the axon. Rab4 associates with early and recycling endosomes carrying a diverse range of proteins. The FRB-FKBP assay indicated that a particular subset of Rab4-only vesicles associates with Kinesin-2. These vesicles predominantly move toward the synapse in axons. Although Kinesin-2 is also found to bind and transport vesicles containing Acetylcholinesterase (AChE) in the same axon, it was observed that Rab4 does not mark them. Similarly, the post-Golgi vesicles carrying N-cadherin and β-catenin, which are known to associate with Kinesin-2, exclude Rab4. Therefore, the Rab4-associated vesicles transported in the axon are likely to contain a unique set of proteins (Dey, 2017).

An association between RabGTPases and putative transport proteins was identified using coimmunoprecipitation, pull-down, and yeast two-hybrid assays. While these assays provide information on the possible interactions, their nature in a cellular or physiological scenario is unknown. Long-range transport of the Rab27/CRMP2 complex by Kinesin-1 and the Rab3/DENN MADD complex by KIF1A/1Bβ are substantiated by genetic perturbations and imaging. The FKBP-FRB-inducible interaction system used in this study identified Kinesin-2α and KIF13A/B as the key motors for nascent Rab4-associated vesicles. The latter was also shown to associate with Rab5-marked early endosomes. The results excluded the possibility that the Rab4 effectors involved in this interaction could bind to Rab5 and Rab11. In vivo analysis of Rab4-associated vesicular traffic further confirmed that heterotrimeric Kinesin-2 plays a dominant role in the trafficking of nascent Rab4 vesicles toward the synapse (Dey, 2017).

Anterograde axonal movement of Rab4 vesicles is mostly mediated by Kinesin-2, which transports nascent Rab4 vesicles, whereas Kinesin-3 transports sorting endosomes. In a recent study, it was shown that combined activity of Kinesin-1 and Kinesin-2 steers AChE-containing vesicles in the axon. A simultaneous association between these two motors was suggested to induce longer runs of AChE-containing vesicles. A similar collaboration between Khc-73 and Kinesin-2 could propel longer runs of Rab4 vesicles. Khc-73 has been implicated in neuronal functions. Khc-73 knockdown in lch5 neurons decreased the motility of Rab4-associated vesicles to a limited extent, which suggests that the motor is only engaged in transporting a relatively small fraction of Rab4-associated vesicles in axons. Khc-73 is a processive motor with an in vitro velocity of 1.54 ± 0.46 &mi;m/s, and this study found that Khc-73 RNAi particularly affected the longer runs. Thus, Khc-73 could support the relatively longer runs (3.05 &mi;m) of Rab4 vesicles in axons. It was previously shown that Rab5-associated vesicles bind to KIF13A/B/Khc-73. Hence, it is inferred that sorting endosomes, positive for both Rab4 and Rab5, can engage both Kinesin-2 and KIF13A/Khc-73 through the cognate adaptors of these two Rabs. However, one needs suitably tagged reagents to establish this conjecture through direct observation in vivo (Dey, 2017).

Local synaptic vesicle recycling mediated by RabGTPases is critical for the availability of fusion-ready synaptic vesicles to sustain neurotransmission. The machinery involved in the translocation of RabGTPases from cell bodies to the synapses is poorly understood. Certain RabGTPases such as Rab3 and Rab27 are known to associate with Kinesin-3 and Kinesin-1, respectively, for their anterograde axonal transport. Rab4 is a protein present on the endosomal sorting intermediates, and it allows for the transfer of cargoes from early endosomes to recycling endosomes. As Rab4 is present in both early and recycling endosomal populations, the logistics of Rab4 transport into the presynaptic terminal has been unclear. The current results show that Rab4-associated vesicles are present throughout the neuron and moderately enriched in the synapse, which is a consequence of a small anterograde bias in their transport (Dey, 2017).

The activity of Rab4 is critical for the physiology of the neuron-like extension of the growth cone. The overexpressed levels and activity of Rab4 found in conjunction with neuropathy, such as Alzheimer's disease, suggest that overactivation of the endosomal system could influence disease progression. In Drosophila, Rab4 is expressed in both neuronal and non-neuronal tissues throughout development with the exception of optic lobe neurons of the pupal brain. These studies project the idea that the expression and activity of Rab4 are crucial for the maintenance of neuronal physiology. This study has shown that the synaptic Rab4 is maintained through axonal transport by heterotrimeric Kinesin-2 and Kinesin-3. As the synaptic localization of Rab4 is dependent on its active form, other factors like PI3K are also implicated in this trafficking. The results further suggest that the function and localization of Rab4 at axon termini are critical for the maintenance of synaptic balance at ventral ganglion. Overactivation of Rab4 in cholinergic neurons reduced the synapse-bearing region of the ventral ganglion, although levels of the marker, Brp, remained unchanged. This observation suggests that local Rab4 activity at the axon termini suppresses synapse formation. Consistent with this conjecture, this study found that the synapse-bearing zone at the neuropil expands when Rab4DN is overexpressed. Although the current data are insufficient to explain the underlying mechanism, they indicate that alteration of the trafficking logistics driven by heterotrimeric Kinesin-2 and Kinesin-3 family motors might play an essential role in the development of the defect. Further analysis of larval behavior and neuronal stability in the ventral ganglion with aging would be useful in uncovering the mechanism (Dey, 2017).

Kinesin-2 and Apc function at dendrite branch points to resolve microtubule collisions

In Drosophila neurons, kinesin-2, EB1 and Apc are required to maintain minus-end-out dendrite microtubule polarity, and it has been proposed that they steer microtubules at branch points. Motor-mediated steering of microtubule plus ends could be accomplished in two ways: 1) by linking a growing microtubule tip to the side of an adjacent microtubule as it navigates the branch point (bundling), or 2) by directing a growing microtubule after a collision with a stable microtubule (collision resolution). Using live imaging to distinguish between these two mechanisms, this study found that reduction of kinesin-2 did not alter the number of microtubules that grew along the edge of the branch points where stable microtubules are found. However, reduction of kinesin-2 or Apc did affect the number of microtubules that slowed down or depolymerized as they encountered the side of the branch opposite to the entry point. These results are consistent with kinesin-2 functioning with Apc to resolve collisions. However, they do not pinpoint stable microtubules as the collision partner as stable microtubules are typically very close to the membrane. To determine whether growing microtubules were steered along stable ones after a collision, the behavior of growing microtubules was analyzed at dendrite crossroads where stable microtubules run through the middle of the branch point. In control neurons, microtubules turned in the middle of the crossroads. However, when kinesin-2 was reduced some microtubules grew straight through the branch point and failed to turn. It is proposed that kinesin-2 functions to steer growing microtubules along stable ones following collisions (Weiner, 2016).

Heterotrimeric Kinesin-2, together with Kinesin-1, steers vesicular acetylcholinesterase movements toward the synapse

Acetylcholinesterase (AChE), which is implicated in the pathophysiology of neurological disorders, is distributed along the axon and enriched at the presynaptic basal lamina. It hydrolyses the neurotransmitter acetylcholine, which inhibits synaptic transmission. Aberrant AChE activity and ectopic axonal accumulation of the enzyme are associated with neurodegenerative disorders, such as Alzheimer's disease. The molecular mechanism that underlies AChE transport is still unclear. This study shows that expression of Drosophila AChE tagged with photoactivatable green fluorescent protein and m-Cherry (GPAC) in cholinergic neurons compensates for the RNA interference-mediated knockdown of endogenous AChE activity. GPAC-AChE, which is enriched in the neuropil region of the brain, moves in the apparently vesicular form in axons with an anterograde bias in Drosophila larvae. Two anterograde motors, Kinesin-1 and -2, propel distinct aspects of GPAC-AChE movements. Total loss of kinesin-2 reduces the density of anterograde traffic and increases bidirectional movements of GPAC-AChE vesicles without altering their speed. A partial loss of kinesin-2 reduces both the density and speed of anterograde GPAC-AChE traffic and enhances the pool of stationary vesicles. Together, these results suggest that combining activity of a relatively weak Kinesin-2 with that of a stronger Kinesin-1 motor could steer AChE-containing vesicles toward synapse, and provides a molecular basis for the observed subcellular distribution of the enzyme (Kulkarni, 2017).

The C-terminal tails of heterotrimeric kinesin-2 motor subunits directly bind to alpha-tubulin1: Possible implications for cilia-specific tubulin entry

The assembly of microtubule-based cytoskeleton propels the cilia and flagella growth. Previous studies have indicated that the kinesin-2 family motors transport tubulin into the cilia through intraflagellar transport. This study reports a direct interaction between the C-terminal tail fragments of heterotrimeric kinesin-2 and alpha-tubulin1 isoforms in vitro. Blot overlay screen, affinity purification from tissue extracts, cosedimentation with subtilisin-treated microtubule and LC-ESI-MS/MS characterization of the tail-fragment-associated tubulin identified an association between the tail domains and alpha-tubulin1A/D isotype. The interaction was confirmed by Forster's resonance energy transfer assay in tissue-cultured cells. The overexpression of the recombinant tails in NIH3T3 cells affected the primary cilia growth, which was rescued by coexpression of a alpha-tubulin1 transgene. Furthermore, fluorescent recovery after photobleach analysis in the olfactory cilia of Drosophila indicated that tubulin is transported in a non-particulate form requiring kinesin-2. These results provide additional new insight into the mechanisms underlying selective tubulin isoform enrichment in the cilia (Girotra, 2017).

Drosophila homolog of human KIF22 at the autism-linked 16p11.2 loci influences synaptic connectivity at larval neuromuscular junctions

Copy number variations at multiple chromosomal loci, including 16p11.2, have been implicated in the pathogenesis of autism spectrum disorder (ASD), a neurodevelopmental disease that affects 1-3% of children worldwide. This study investigated the roles of human genes at the 16p11.2 loci in synaptic development using Drosophila larval neuromuscular junctions (NMJ), a well-established model synapse with stereotypic innervation patterns. A preliminary genetic screen based on RNA interference was conducted in combination with the GAL4-UAS system, followed by mutational analyses. Data indicate that disruption of klp68D, a gene closely related to human KIF22, causes ectopic innervations of axon branches forming type III boutons in muscle 13, along with less frequent re-routing of other axon branches. In addition, mutations in klp64D, of which gene product forms Kinesin-2 complex with KLP68D, leads to similar targeting errors of type III axons. Mutant phenotypes are at least partially reproduced by knockdown of each gene via RNA interference. Taken together, these data suggest the roles of Kinesin-2 proteins, including KLP68D and KLP64D, in ensuring proper synaptic wiring (Park, 2016).

Recent clinical studies on ASD have revealed gross alterations in the structure of nervous system. For instance, total brain size and the rate of neuronal proliferation in the prefrontal cortex are significantly increased in ASD patients. Such structural changes may reflect altered neuronal connectivity between specific brain regions. Moreover, the proposed candidate genes responsible for ASD include various synaptic proteins that play important roles in neurite outgrowth, axonal guidance, axonal targeting and synaptogenesis, suggesting structural abnormalities at a synaptic level responsible for expression of ASD phenotypes. Based on these findings, it was hypothesized that genetic perturbation at the 16p11.2 loci would lead to aberrant synaptic connectivity, thus underlying functional disturbances that lead to ASD. Results demonstrate significant axon targeting errors caused by defects in KLP68D, a Drosophila Kinesin-2 protein closely related to human KIF22 at the autism-linked 16p11.2 loci (Park, 2016).

Hetero-trimeric Kinesin-2 complex in Drosophila, consisting of KLP68D, KLP64D and DmKAP, has been implicated in microtubule organization and axonal transport of synaptic proteins such as choline acetyltransferase. However, experimental evidence is missing to support the idea that Kinesin-2 complex may participate in delivering molecules important for axon targeting. Potential cargos of KLP68D and KLP64D motors have been estimated to include Unc-51/ATG1, Fasciclin II, EB1, Armadillo, Bazooka, and DE-cadherin, most of whom have been well characterized for their roles in synaptogenesis. It will be important to investigate whether disruptions of any of these potential cargos lead to aberrant axon targeting phenotypes observed in Klp68D and Klp64D mutants (Park, 2016).

It should be noted that Drosophila Nod ('no distributive disjunction'), mostly involved in chromosomal segregation has been recognized as a homolog for human KIF22. However, similar levels of sequence homology to KIF22 were found in both Nod and KLP68D. In fact, a blast analysis results in higher sequence identity between KIF22 and KLP68D than Nod (41% vs. 33%). The specificity of motor protein cargos is often predicted to depend on the amino acid composition of motor proteins outside their core motor domain. Therefore, relatively lower level of homology between human KIF22 and Drosophila KLP68D may correspond to their distinct molecular functions. In contrast to KLP68D, the role of KIF22 in the mammalian nervous system has not been extensively investigated, but only limited to chromosomal segregation and genomic stability. Whether Drosophila KLP68D can be functionally replaced by human KIF22 in transgenic animals awaits further investigations (Park, 2016).

Kinesin-II recruits Armadillo and Dishevelled for Wingless signaling in Drosophila

Wingless (Wg)/Wnt signaling is fundamental in metazoan development. Armadillo (Arm)/beta-catenin and Dishevelled (Dsh) are key components of Wnt signal transduction. Recent studies suggest that intracellular trafficking of Wnt signaling components is important, but underlying mechanisms are not well known. This study shows that Klp64D, the Drosophila homolog of Kif3A kinesin II subunit, is required for Wg signaling by regulating Arm during wing development. Mutations in klp64D or RNAi cause wing notching and loss of Wg target gene expression. The wing notching phenotype by Klp64D knockdown is suppressed by activated Arm but not by Dsh, suggesting that Klp64D is required for Arm function. Furthermore, klp64D and arm mutants show synergistic genetic interaction. Consistent with this genetic interaction, Klp64D directly binds to the Arm repeat domain of Arm and can recruit Dsh in the presence of Arm. Overexpression of Klp64D mutated in the motor domain causes dominant wing notching, indicating the importance of the motor activity. Klp64D shows subcellular localization to intracellular vesicles overlapping with Arm and Dsh. In klp64D mutants, Arm is abnormally accumulated in vesicular structures including Golgi, suggesting that intracellular trafficking of Arm is affected. Human KIF3A can also bind β-catenin and rescue klp64D RNAi phenotypes. Taken together, it is proposed that Klp64D is essential for Wg signaling by trafficking of Arm via the formation of a conserved complex with Arm (Vuong, 2014).

Interaction with a kinesin-2 tail propels choline acetyltransferase flow towards synapse

Bulk flow constitutes a substantial part of the slow transport of soluble proteins in axons. Though the underlying mechanism is unclear, evidences indicate that intermittent, kinesin-based movement of large protein-aggregates aids this process. Choline acetyltransferase (ChAT), a soluble enzyme catalyzing acetylcholine synthesis, propagates toward the synapse at an intermediate, slow rate. The presynaptic enrichment of ChAT requires heterotrimeric kinesin-2, comprising KLP64D, KLP68D and DmKAP, in Drosophila. This study shows that the bulk flow of a recombinant Green Fluorescent Protein-tagged ChAT (GFP::ChAT), in Drosophila axons, lacks particulate features. It occurs for a brief period during the larval stages. In addition, both the endogenous ChAT and GFP::ChAT directly bind to the KLP64D tail, which is essential for the GFP::ChAT entry and anterograde flow in axon. These evidences suggest that a direct interaction with motor proteins could regulate the bulk flow of soluble proteins, and thus establish their asymmetric distribution (Sadananda, 2012).

Kinesin II is required for cell survival and adherens junction positioning in Drosophila photoreceptors

Photoreceptor morphogenesis requires specific and coordinated localization of junctional markers at different stages of development. This study provides evidence that Drosophila Klp64D, a homolog of Kif3A motor subunit of the heterotrimeric Kinesin II complex, is essential for viability of developing photoreceptors and localization of junctional proteins. Genetic analysis of mutant clones shows that absence of Klp64D protein in early larval eye disc does not affect initial differentiation, but results in abnormal nuclear position in differentiating photoreceptors. These cells eventually die in the pupal stage, indicating klp64D's role in cell viability. The function of Klp64D protein is cell type specific because the p35 cell death inhibitor can rescue cell death in cone cells but not photoreceptors. In contrast to early induction of mutant clones, late induction during third instar larval stage just prior to pupation allows survival of single- or few-celled clones of klp64D mutant cells. Analysis of these lately induced clones shows that Klp64D function is essential for Bazooka (Par-3 homolog) and Armadillo localization to the adherens junction (AJ) in pupal photoreceptors. These findings suggest that Kinesin II complex plays a cell type-specific function in the localization of AJ and cell polarity proteins in the developing retina, thereby contributing to photoreceptor morphogenesis (Mukhopadhyay, 2010).

Directed microtubule growth, +TIPs, and kinesin-2 are required for uniform microtubule polarity in dendrites

In many differentiated cells, microtubules are organized into polarized noncentrosomal arrays, yet few mechanisms that control these arrays have been identified. For example, mechanisms that maintain microtubule polarity in the face of constant remodeling by dynamic instability are not known. Drosophila neurons contain uniform-polarity minus-end-out microtubules in dendrites, which are often highly branched. Because undirected microtubule growth through dendrite branch points jeopardizes uniform microtubule polarity, this study used this system to understand how cells can maintain dynamic arrays of polarized microtubules. Growing microtubules navigate dendrite branch points by turning the same way, toward the cell body, 98% of the time, and growing microtubules track along stable microtubules toward their plus ends. Using RNAi and genetic approaches, this study shows that kinesin-2, and the +TIPS EB1 and APC, are required for uniform dendrite microtubule polarity. Moreover, the protein-protein interactions and localization of Apc2-GFP and Apc-RFP to branch points suggests that these proteins work together at dendrite branches. The functional importance of this polarity mechanism is demonstrated by the failure of neurons with reduced kinesin-2 to regenerate an axon from a dendrite. It is concluded that microtubule growth is directed at dendrite branch points and that kinesin-2, APC, and EB1 are likely to play a role in this process. It is propose that Kinesin-2 is recruited to growing microtubules by +TIPS and that the motor protein steers growing microtubules at branch points. This represents a newly discovered mechanism for maintaining polarized arrays of microtubules (Mattie, 2010).

Most cells in multicellular organisms contain polarized noncentrosomal microtubule arrays. In interphase mammalian cultured cells, microtubules are nucleated at the centrosomal microtubule organizing center (MTOC), and plus ends grow towards the cell periphery. However, in many differentiated cells, minus ends are not focused at a centrosomal MTOC. In epithelial cells, a major population of microtubules has minus ends focused at the apical side and plus ends at the basal side. In muscle cells, minus ends spread out around the nuclear envelope, and neurons have perhaps the simplest and most strikingly polarized noncentrosomal microtubule arrays. The mechanisms that organize these noncentrosomal microtubule arrays are poorly understood (Mattie, 2010).

Neurons have two types of processes that extend from the cell body: axons and dendrites. Dendrites primarily receive signals from other neurons or the environment, and axons send signals to other neurons or output cells. One basic difference between axons and dendrites is the arrangement of microtubules. In axons microtubules are arranged into an overlapping array of uniform polarity plus-end-out microtubules. In dendrites of cultured mammalian neurons microtubules have mixed orientation near the cell body. In dendrites of Drosophila neurons 90%-95% of microtubules have minus ends distal to the cell body. As the dendritic array in Drosophila is very simple, and extremely different from a centrosomal array, This study used it as a model system to identify mechanisms that organize polarized noncentrosomal microtubules (Mattie, 2010).

It is not known how uniform dendrite microtubule polarity is established or maintained. Models for generating the plus-end-out axonal microtubule array focus on sliding of microtubules by motor proteins. In mammalian neurons, microtubules are thought to be nucleated in the cell body at the centrosome, then released from the centrosome and transported down the axon in the correct orientation by motors including dynein. Models to account for mixed orientation microtubules in dendrites of cultured neurons have also been proposed. In this case, the kinesin MKLP1 (Kif23) has been proposed to transport minus-end-out microtubules into dendrites along plus-end-out microtubules. The current study identified a new mechanism that is required for uniform microtubule polarity in dendrites (see Interactions between kinesin-2 and +TIPs, and localization of Apc2-GFP to dendrite branch points). As it uses conserved, generally expressed proteins, it could play a role in maintaining microtubule polarity in many other cell types (Mattie, 2010).

Using Drosophila dendrites as a model system, this study demonstrates that growing microtubule plus ends almost always turn towards the cell body at branch points, and that they track stable microtubules through branches. Kinesin-2, EB1 and APC are all required to maintain microtubule polarity and are linked in an interaction network. Based on these results, a model is proposed for directed growth of microtubules in dendrites. Apc2 most likely contains a branch localization signal because Apc2-GFP localizes well to dendrite branches even when overexpressed. Localization of Apc2 to dendrite branch points can recruit Apc to the branch. Apc can interact with the Kap3 subunit of kinesin-2, and so could increase concentration of the motor near the branch. EB1-GFP does not concentrate at branches, so it is proposed that a growing microtubule plus end coated with EB1 is transiently linked to kinesin-2 as it passes through the branch, through the interaction between Apc and the EB1 tail. SxIP motifs in Apc and Klp68D could also contribute to this interaction. As both Kap3 and the SxIP motif in Klp68D are in the kinesin-2 tail, the motor domain would be free to walk along a nearby stable microtubule towards the plus end and cell body (Mattie, 2010).

Even a very brief application of force pulling the growing microtubule towards the cell body should be sufficient to steer growth towards the cell body. Once the tip of the microtubule turns, growth would be constrained by the dendrite walls. The association of the growing plus end with stable microtubule would probably only need to be maintained over a distance of a micron. This model is consistent with the observations that kinesin-2 has shorter run lengths than kinesin-1, and that individual EB1 interactions with the microtubule plus end persist less than a second (Mattie, 2010).

Observations of plus end behavior in vivo also favor a model in which only transient interactions of the motor and microtubule plus end are involved, because plus ends turning sharply are frequently seen. Stable microtubules do not accommodate such sharp turns, so the sharp turns of plus ends are not likely to occur while the plus end is tracking along a stable microtubule. Instead they likely represent a switch from a freely growing plus end to one that is following a track (Mattie, 2010).

Directed growth of microtubules at dendrite branch points allows dendrites to maintain uniform minus-end-out polarity despite continued microtubule remodeling. This mechanism is also likely necessary to establish uniform microtubule polarity in branched dendrites, but probably cannot account for minus-end-out polarity on its own. It is hypothesized that directed microtubule growth is used in concert with an unidentified mechanism to control microtubule polarity in dendrites. Transport of oriented microtubule pieces has been proposed to contribute to axon microtubule polarity and a similar mechanism could play a role in dendrites. For example, a kinesin anchored to the cell cortex by its C-terminus could shuttle minus-end-out microtubule seeds into dendrites. Alternately, polarized microtubule nucleation sites could be localized within dendrites. Identifying directed growth as one mechanism that contributes to dendrite microtubule polarity should facilitate identification of the missing pieces of this puzzle (Mattie, 2010).

Because kinesin-2, APC, EB1 and polarized microtubules are found in many cell types, directed growth of microtubules along stable microtubule tracks could be very broadly used for maintaining microtubule polarity. This is a newly identified function for both the +TIP proteins and kinesin-2. APC has previously been localized to junctions between a microtubule tip and the side of another microtubule at the cortex of epithelial cells, and sliding of microtubule tips along the side of microtubules was also seen in this system, but there was no association with a particular direction of movement or overall microtubule polarity (Mattie, 2010).

As kinesin-2 has previously been shown to be enriched in tips of growing axons in cultured mammalian neurons, as has APC, it is possible that they could mediate tracking of growing microtubules along existing microtubule tracks in the growth cone. In fact, this type of microtubule tracking behavior has been observed in axonal growth cones under low actin conditions. Thus directed growth of microtubules could also be important during axon outgrowth. Indeed, the same principle of directed growth by a motor protein connected to a growing microtubule plus end could be used to align microtubules in many circumstances (Mattie, 2010).

Kinesin-2 differentially regulates the anterograde axonal transports of acetylcholinesterase and choline acetyltransferase in Drosophila

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) are involved in acetylcholine synthesis and degradation at pre- and postsynaptic compartments, respectively. This study shows that their anterograde transport in Drosophila larval ganglion is microtubule-dependent and occurs in two different time profiles. AChE transport is constitutive while that of ChAT occurs in a brief pulse during third instar larva stage. Mutations in the kinesin-2 motor subunit Klp64D and separate siRNA-mediated knock-outs of all the three kinesin-2 subunits disrupt the ChAT and AChE transports, and these antigens accumulate in discrete nonoverlapping punctae in neuronal cell bodies and axons. Quantification analysis further showed that mutations in Klp64D could independently affect the anterograde transport of AChE even before that of ChAT. Finally, ChAT and AChE were coimmunoprecipitated with the kinesin-2 subunits but not with each other. Altogether, these suggest that kinesin-2 independently transports AChE and ChAT within the same axon. It also implies that cargo availability could regulate the rate and frequency of transports by kinesin motors (Baqri, 2006).

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-bound 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. Kap mutants were generated and it was shown 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 (Pflanz, 2004).

In Drosophila, Kinesin II forms a heterotrimeric complex composed of two motor subunits, KLP68D and KLP64D, and a singular nonmotor protein, the Kinesin-associated protein 3 (Kap3), which acts as adaptor and regulator of the complex (Ray, 1999). Recent studies on KLP64D mutants suggest that Kinesin II is specifically required for the axonal transport of choline acetyltransferase in a subgroup of neurons (Ray, 1999). A similar phenotype was reported for Kap mutants (Sarpal, 2003), suggesting that Kap/Kinesin II complexes are only required for neural function. However, in mice, humans and rats as well as in sea urchins and algae, Kinesin II was shown to be necessary for the formation and maintenance of cilia/axoneme structures. In some organisms, a third motor protein, Kif3C, has been identified. Kif3C was proposed to be part of distinct Kinesin II complexes, which act in a spatiotemporal and/or cell-specific fashion. A Kif3C homolog has been identified in the Drosophila genome (CG17461), suggesting that the previous studies may not have revealed all aspects of Kinesin II-dependent Kap function in the fly (Ray, 1999; Sarpal, 2003). To test this possibility, Kap lack-of-function mutants were generated. In addition to the recently described neural function, Kap activity is also required in germ cells for proper follicle separation during oogenesis. The results suggest that Kap participates in signalling necessary for the establishment of follicle-separating stalk cells (Pflanz, 2004).

To assess Kinesin II requirement, mutations affecting the single non-motor component Kap of Drosophila were generated. A semilethal mutation, l(1)G0396, was recovered in which a P{lacW} element was inserted in the first intron of the Kap gene. In all, 95% of hemizygous l(1)G0396 males develop into pupae and die as pharate adults; about 5% hatch and show a paralytic phenotype as described for KLP64D (Ray, 1999). Precise excisions of the P-element resulted in wild-type flies, indicating that the P insertion is the cause of the mutation. Imprecise excisions were also made that represent hypomorphic Kap alleles, which have small internal deletions in the protein coding sequence. In addition, alleles such as Kap89 were obtained that lack the promoter and the first exon of the gene. Homozygous Kap89 mutants fail to express Kap as revealed by in situ hybridization. In addition, their phenotype was as strong as the phenotype of transheterozygous Kap89/Df(1)v-N48 mutant individuals. These findings indicate that Kap89 is a null mutation. Kap89 and the hypomorphic mutants could be rescued by Kap activity that was derived from an Actin5C enhancer-driven Kap cDNA transgene, confirming that the mutations affect only the Kap gene (Pflanz, 2004).

To determine the sites of Kap expression in the organism, RNA in situ hybridization was performed on staged ovaries and embryos. During oogenesis, Kap expression is observed in nurse cells from where transcripts are transported into the growing oocyte. The transcripts remain ubiquitously distributed in eggs and embryos until the blastoderm stage. Zygotic Kap expression is initiated during gastrulation in both ectoderm and mesoderm and is subsequently enriched in neurons. Based on the strong maternal expression of the gene, it was asked whether Kap also has a role during oogenesis in addition to its recently reported function in the nervous system (Pflanz, 2004).

To assess the function of maternal Kap activity, homozygous Kap mutant germline clones were generated using the FLP/ovoD1 system. Females with homozygous Kap89 mutant germ lines are sterile; germline mutant follicles degenerate after they reached stage 6 of oogenesis. Up to this stage, mutant follicles lacking Kap contain more cells than wild-type follicles. The supernumerary cells are either only nurse cells (type I follicles) or both nurse cells and oocytes (type II follicles). Type II follicles have multiple oocytes and a corresponding number (ratio 1:15) of extra nurse cells. Of all follicles scored, 50% showed mixed type I/II follicles. Of the remaining follicles, few (~5%) showed either type I or type II follicles only (Pflanz, 2004).

The supernumerary cells in type I follicles suggest that the mutant germline cells have undergone more than the normal four rounds of mitotic divisions. To confirm this proposal, the number of ring channels and the fusome of the germline mutant ovaries were inspected. Both structures derive from an incomplete separation of daughter cells after the division, resulting in a maximum of four ring channels in the case of the wild-type oocyte and in a branched fusome structure that interconnects the germ cells of a follicle. Kap mutant germ cells were found that have more than four ring channels and fusomes connecting up to about 50 cells. This indicates that at least one extra round of mitotic divisions occurs in Kap mutant follicles. The use of oocyte markers, such as gurken (grk) and oskar (osk) mRNA, identified either one oocyte or multiple oocytes in ectopic locations within the follicles. Collectively, these findings indicate that the determination and the initial stages of oocyte differentiation do not depend on Kap activity, whereas the cellular processes underlying the formation of individual follicles and regulation of germ cell proliferation do (Pflanz, 2004).

Kinesin I has previously been shown to participate in the transport of distinct mRNA species and associated protein to the posterior pole of the Drosophila oocyte. To assess the integrity of this type of microtubule-dependent transport in Kap89 mutant follicles, expression and localization of the oo18 RNA-binding protein (Orb) were examined. Orb is necessary for the directed transport and localized translation of grk and osk mRNA, the axial polarity determinants of the growing oocyte. The expression of Orb is initiated normally, although it is not maintained subsequently. The oocyte determination factor Bic-D and ß-tubulin are expressed normally, and oskar RNA is normally expressed and transported in Kap89 mutant follicles. Thus, cytoskeletal structures and the transport of oskar mRNA into the growing oocyte appear not to be directly affected by the lack of Kap activity (Pflanz, 2004).

Separation of follicles during early oogenesis involves the formation of a characteristic intervening stalk structure. This structure derives from a small group of precursor cells (located in germarium region 2B), which develop into polar follicle cells and the stalk cells. Their proper development depends on cell-cell communication events between the germline and soma cells as shown by Notch/Delta, JAK/STAT and Hedgehog signalling mutants, which develop fused follicles and lack morphologically distinct stalks. Since such a phenotype was also observed with the Kap mutant ovaries, it was asked whether polar follicle cells (which can be visualized by A101-lacZ expression) and stalk cells are formed. In Kap mutant follicles, supernumerary A101-lacZ expressing polar follicle cells were observed, whereas follicle-separating stalk cells were absent. Identical results were obtained by anti-FasIII antibody staining, a different marker for polar follicle cells. These observations suggest that stalk precursor cells fail to differentiate, remain in the follicle epithelium and express the molecular characteristics of follicle cells as has been observed in Notch mutants. Clonal analysis shows that stalk cells are formed properly in the absence of Kap activity in the somatic epithelial cells. Thus, Kap activity has a cell autonomous effect on germline cell proliferation, and the absence of Kap in germ cells also affects somatic precursor cells in a non-cell autonomous manner (Pflanz, 2004).

The non-autonomous Kap effect on follicle cell determination could be explained by a requirement for Kap for the transport and/or localization of components that are needed to signal cell fate to the surrounding somatic precursor cells. Therefore Kap mutant ovaries were examined for the expression of Delta, which activates Notch signalling, hedgehog and the JAK/STAT-activating ligand unpaired. Delta and hedgehog expressions were not affected, whereas unpaired failed to be expressed in polar follicle cells of Kap germline mutant ovaries. This observation suggests that lack of Kap activity interferes with the signalling-mediated crosstalk between germline and somatic cells (Pflanz, 2004).

These results provide evidence that Kap activity in germline cells is required for the proper differentiation of a distinct group of epithelial cells, the stalk cells, which separate individual follicles during early oogenesis. The absence of Kap in germ cells prevents somatic target cells from differentiating into proper polar follicle cells, because they fail to express the ligand Unpaired. Unpaired activates JAK/STAT signalling and thereby induces stalk cell fate in the respective precursor cells. The details of the germline cell/polar follicle cell/stalk cell signalling cascade are currently being investigated to address the cellular mechanism of these interactions (Pflanz, 2004).

Decoding cilia function: defining specialized genes required for compartmentalized cilia biogenesis

The evolution of the ancestral eukaryotic flagellum is an example of a cellular organelle that became dispensable in some modern eukaryotes while remaining an essential motile and sensory apparatus in others. To help define the repertoire of specialized proteins needed for the formation and function of cilia (see Kimball's Cilia and Flagella site or UTMB Cell Biology Topics), comparative genomics was used to analyze the genomes of organisms with prototypical cilia, modified cilia, or no cilia and 200 genes were identified that are absent in the genomes of nonciliated eukaryotes but are conserved in ciliated organisms. Importantly, over 80% of the known ancestral proteins involved in cilia function are included in this small collection. Using Drosophila as a model system, a novel family of proteins (OSEGs: outer segment) was then characterized that is essential for ciliogenesis. Osegs encode components of a specialized transport pathway unique to the cilia compartment and are related to prototypical intracellular transport proteins (Avidor-Reiss, 2004).

The bioinformatics approach also identified two kinesin II subunits as cilia-compartment genes. Kinesin II has been shown to be required for cilia assembly in a variety of organisms and was proposed to function as the anterograde motor carrying cargo from the base of the cilia to its distal tip. If OSEGs mediate the kinesin-based intraciliary transport, and if this transport were specifically required for outer segment formation, it was reasoned that mutations in klp64D, the central component of Drosophila kinesin II, should generate in vivo phenotypes that resemble oseg defects. Thus, flies were generated defective in klp64D function and mechano- and chemo-sensory physiology and the transport and accumulation of α1tub84B into sensory cilia were examined. klp64D mutant animals share all of the hallmarks of oseg2 mutants: (1) severe chemoinsensitivity, (2) a total loss of mechanoreceptor currents, (3) GFP-α1tub84B completely failing to enter the outer segments, and (4) microtubules dramatically accumulating at the base of the cilia. Furthermore, klp64D animals, just like oseg2 mutants, have an almost complete loss of the tubular body, but have normal basal bodies and connecting cilia; thus, kinesin II is also not essential for the assembly of the proximal ciliary structures, including axoneme components. Together, these results substantiate kinesin II as a critical player in OSEG function and validate the fundamental importance of intraciliary transport in outer segment (compartmentalized cilia) biogenesis (Avidor-Reiss, 2004).

Drosophila KAP interacts with the kinesin II motor subunit KLP64D to assemble chordotonal sensory cilia, but not sperm tails

Kinesin II-mediated anterograde intraflagellar transport (IFT) is essential for the assembly and maintenance of flagella and cilia in various cell types. Kinesin associated protein (KAP) is identified as the non-motor accessory subunit of Kinesin II, but its role in the corresponding motor function has not been characterized. Mutations in the Drosophila KAP (DmKap) gene eliminate the sensory cilia as well as the sound-evoked potentials of Johnston's organ (JO) neurons. Ultrastructure analysis of these mutants reveals that the ciliary axonemes are absent. Mutations in Klp64D, which codes for a Kinesin II motor subunit in Drosophila, show similar ciliary defects. All these defects are rescued by exclusive expression of DmKAP and KLP64D/KIF3A in the JO neurons of respective mutants. Furthermore, reduced copy number of the DmKap gene was found to enhance the defects of hypomorphic Klp64D alleles. Unexpectedly, however, both the DmKap and the Klp64D mutant adults produce vigorously motile sperm with normal axonemes. It is concluded that KAP plays an essential role in Kinesin II function, which is required for the axoneme growth and maintenance of the cilia in Drosophila type I sensory neurons. However, the flagellar assembly in Drosophila spermatids does not require Kinesin II and is independent of IFT (Sarpal, 2003).

The type I sense organs of Drosophila, namely, the chordotonal organs, the mechanosensory bristles, and the taste and olfactory sensilla, are innervated by bipolar sensory neurons, each with a single dendritic cilium containing 9+0 axonemal organization of microtubules. Each chordotonal organ neuron has a single long cilium, the assembly of which begins from the distal basal bodies in the dendrite; each cilium is attached to a tube-shaped dendritic cap at the apex. [Type I external mechanosensory bristles include three support cells and one neuron. The innermost support cell (sheath cell) forms a tubular extension around the sensory process and produces an extracellular dendritic cap that covers the cilium tip]. Such chordotonal organs are found in the second antennal segment, where they are required for hearing and are termed Johnston's organ (JO), and in various other parts of the body, where they are required for proprioception. Mutants with auditory defects have defective dendritic cilia in JO neurons. The mechanisms that form and maintain such ciliary structures are only beginning to be elucidated (Sarpal, 2003 and references therein).

Extensive studies in Chlamydomonas and other organisms have shown that flagellar and ciliary proteins are synthesized in the cell body and are then transported in preassembled IFT complexes to the distal tip of the flagella by a mechanism called 'intraflagellar transport' (IFT). This process is essential for the assembly and maintenance (via turnover) of flagella. Members of the Kinesin II family of motor proteins and cytoplasmic Dynein motors are known to play important roles in IFT (Sarpal, 2003 and references therein).

Kinesin II holoenzyme was purified from sea urchin embryos as a heterotrimer of two dissimilar motor subunits and a third non-motor accessory subunit called 'kinesin-associated protein'. The motor subunits contain a globular plus end-directed, microtubule-dependent ATPase domain at the N terminus, and they associate with each other via a coiled-coil stalk domain in the middle. The KAP subunit is estimated to bind to the C-terminal tail domains of the motor subunits (Marszalek, 2000b). Support for the trimeric composition of Kinesin II has been provided in various vertebrate and invertebrate organisms, and this motor has been implicated in a variety of intracellular transport processes in vivo (Sarpal, 2003 and references therein).

Kinesin II is the motor for the anterograde IFT, and studies with different types of ciliated cells from Chlamydomonas to humans have shown that it is essential for ciliogenesis as well as for flagellar growth and maintenance. For example, conditional mutations in the fla10 gene of Chlamydomonas block anterograde IFT in the flagella when grown at nonpermissive temperatures. As a result, the flagella gradually reduce and eventually disappear. FLA10 is homologous to the Kinesin II motor subunit, which is associated with the IFT complex subunits in the flagella. Disruption of Kinesin II activity in sea urchin cilia and Tetrahymena flagella are also shown to affect axonemal assembly. Similarly, mutations in the osm-3 locus of Caenorhabditis elegans cause defective chemotaxis behavior, and the distal segments of the dendritic cilia of the chemosensory neurons are absent. These studies suggested that Kinesin II is required for the transport of essential ciliary components in these neurons. In addition, mouse KIF3A and KIF3B play an important role in the assembly of motile cilia in embryonic nodal cells. KIF3A is also localized to the connecting cilia of photoreceptor neurons in the retina, which have 9+0 organization of microtubules in the axoneme, and KIF3A has been implicated in the transport of opsin and arrestin to the outer photoreceptor compartment. Although these pieces of evidence strongly indicate that Kinesin II is a good candidate for the transport of components required for the assembly and maintenance of eukaryotic cilia and flagella, little is known about the role of KAP in this process. An in vivo analysis in C. elegans with GFP-tagged OSM-6 and KAP has shown that the two proteins transport along the sensory cilium at a rate similar to the in vitro rate of Kinesin II; this finding indicates that KAP is associated with the IFT complex in the cilium (Sarpal, 2003 and references therein).

KLP64D, KLP68D, and DmKAP are predicted to form the Kinesin II holoenzyme in Drosophila, and they are shown to coexpress in ciliated sensory neurons during embryogenesis (Pesavento, 1994; Ray, 1999; Sarpal, 2002). The expression levels are particularly high in the neurons innervating the lateral chordotonal organs as well as the anterior sense organs (Ray, 1999; Sarpal, 2002), and these have well-defined dendritic cilia. This indicates that Drosophila Kinesin II could play an important role in ciliogenesis. Therefore, to define a functional assay to study the role of KAP and other Kinesin II-associated proteins in ciliogenesis, auditory responses of Drosophila carrying mutations in the DmKap and Klp64D genes were studied. This revealed that KAP plays a critical role in Kinesin II function during ciliogenesis in these type I sensory neurons of Drosophila, and the genetic interaction study suggests that DmKAP interacts with a Kinesin II motor subunit in vivo (Sarpal, 2003).

Other studies have shown that vertebrate homologs of KAP protein associate with the Kinesin II motor subunits, which are implicated in ciliogenesis in various cell types. However, the precise role of KAP in this process has been unknown. Mutations in the DmKap locus are shown to be haplo-insufficient in Klp64D hypomorphic backgrounds and enhance both the auditory reception defects as well as the ciliogenesis defects of the Klp64D alleles. This study establishes that KAP plays a critical role in Kinesin II motor function in vivo. A recent study has further shown that mutations in the nompB locus of Drosophila, which encodes an IFT88/Tg737/OSM-5 homologous protein, also affect auditory responses of JO neurons and that mutations in Klp64D reduce the GFP-NOMPB localization in the cilia. Therefore, the auditory system of Drosophila can be used to further study in vivo interactions between various IFT components (Sarpal, 2003).

Studies in Chlamydomonas have established that the Kinesin II motor subunits associate with a soluble, protein-rich IFT complex, which they transport toward the distal ends of flagella. This anterograde IFT seems to play a critical role in maintaining the flagellar length and activity. The electron microscopic data presented in this study show that both DmKap and Klp64D gene functions are critical for proper axoneme growth in the dendritic cilia of the JO neurons. This suggests that Kinesin II may transport essential axonemal components into the dendritic cilia for the growth and maintenance of the axoneme structure. Thus, Kinesin II activity in the sensory cilia of Drosophila and in the motile cilia and flagella of other organisms appears to be conserved. In contrast, the spermatogenesis in Drosophila seems to be independent of anterograde IFT. This indicates the presence of hitherto unknown mechanisms of axonemal assembly operating in constructing these unusually long flagella (Sarpal, 2003).

It is concluded that DmKap interacts with Klp64D, and these two gene products are involved in axonemal assembly in the sensory cilia of JO neurons, but not in sperm. The genetic interaction study suggests that DmKAP plays an important role in Kinesin II motor activity in vivo. This work has established a genetic interaction paradigm to further study the in vivo functions of Kinesin II and IFT proteins by using auditory function as an assay (Sarpal, 2003).

Dynamic expression of kinesin accessory protein in Drosophila

The Drosophila homolog of the non-motor accessory subunit of kinesin-II motor complex has been identified. It is homologous to the SpKAP115 of the sea urchin, KAP3A and KAP3B of the mouse, and SMAP protein in humans. In situ hybridization using a DmKAP specific cRNA probe has revealed a dynamic pattern of expression in the developing nervous system. The staining first appears in a subset of cells in the embryonic central nervous system at stage 13 and continues till the first instar larva stage. At the third instar larva stage the staining gets restricted to a few cells in the optic lobe and in the ventral ganglion region. It has also stained a subset of sensory neurons from late stage 13 and till the first instar larva stage. The DmKAP expression pattern in the nervous system corresponds well with that of Klp64D and Klp68D as reported earlier. In addition, the DmKAP gene is constitutively expressed in the germline cells and in follicle cells during oogenesis. These cells are also stained using an antibody to KLP68D protein, but mRNA in situ hybridization using KLP64D specific probe has not stained these cells. Together these results proved a basis for further analysis of tissue specific function of DmKAP in future (Sarpal, 2002).

Zygotic DmKAP expression begins at stage 6 when a high level of staining is observed in the cells around the morphogenetic furrows and in the neuro-ectoderm cells at this stage. The staining gradually condenses in a subset of cells in the CNS and these cells appear in every segment in the presumptive ventral ganglion (VNG) region of the developing brain. This staining persists until the end of embryogenesis and here a strong staining is observed in the brain lobes and the ventral ganglion. The DmKAP staining also appears in a subset of sensory neurons in the peripheral nervous system (PNS) at stage 13, and continues till late stage 17 when the embryo is completely developed. At this stage expression was found in (1) the lateral chordotonal organs, (2) Bolwig's organ, (3) and other anterior sense organs. In addition, there is staining in certain sensory neurons of the ventral and dorsal clusters of the chordotonal organ. The pattern of sensory neurons stained by DmKAP probe is identical to that of KLP68D and KLP64D (Sarpal, 2002).

The DmKAP expression in the CNS is found to continue to the first instar larva stage when a strong staining is observed in most of the ventral ganglion and brain lobe cells. In the third instar larva stage the staining is restricted to a few cells in the presumptive optic lobe region while a weak staining is visible in the ventral cortical region. DmKAP positive cells in the optic lobe are present at the insertion site of the optic stalk. This region is known to have cell bodies of cholinergic interneurons. A similar pattern of staining has been seen with a KLP64D specific probe. These data suggests that DmKAP could act in association with KLP64D and KLP68D in the neurons of CNS and PNS (Sarpal, 2002).

This hypothesis is based on co-localization of the respective gene expression in different neurons. Some direct experimental evidence would be necessary to establish this hypothesis. In addition to the nervous system cells, the epithelial cells of the imaginal disc peripodium are also stained at the third instar larva stage. This indicates that DmKAP may play a role in imaginal disc development as well. DmKAP in the disc epithelial cells may function in association with other kinesin-II components or it may function independent of KLP64D and KLP68D. This can be resolved by studying the corresponding expression pattern of these two genes in the imaginal disc and other non-neural tissue (Sarpal, 2002).

Since KLP64D and KLP68D have dissimilar levels of expression in the ovary, the in situ hybridization pattern in this tissue was analyzed using both the DmKAP cRNA and KLP64D cDNA probes, respectively. In addition, KLP68D localization was examined in this tissue using a purified antibody. RNA in situ hybridization data has revealed that DmKAP is expressed in follicle cells and in nurse cells at all stages of oogenesis. There is, however, no staining in other somatic tissue like the stalk cells that links individual vitellarium, and cells of the terminal filament of an ovariole. DmKAP mRNA is also present in stem cells and in all developing cyst cells inside germarium. The staining in nurse cells and in follicle cells persists throughout development. However, the level of expression in nurse cells is elevated from stage 10b. This is likely to contribute to the maternal component of the oocyte. DmKAP gene expression is also observed in all follicle cells that cover the egg chambers at this stage. This data suggests that DmKAP may play an important role during oogenesis. In contrast, the KLP64D cDNA probe revealed no staining during the early developing stages of oogenesis. Only the nurse cells of stage 10b egg chamber stained for KLP64D mRNA and this is likely to contribute as maternal component to oocyte. The KLP64D cDNA probe stains in an exactly identical pattern in the embryos. Furthermore, both the DmKAP cRNA and KLP64D cDNA probes stained in an identical pattern in the embryonic and larval nervous system. This suggests the DmKAP and KLP64D staining patterns in the ovary are a true reflection of tissue specific expressions of the respective genes. It is therefore concluded that the KLP64D does not express itself during early stages of oogenesis (Sarpal, 2002).

KLP68D antigen is present in all the germ line cells in the ovary in a pattern very similar to that of DmKAP mRNA. This suggests that KLP68D and DmKAP genes are simultaneously expressed in a subset of neurons and in all germline cells. Further, genetic analysis using mutants in the KLP68D and DmKAP gene would reveal their functions in the respective tissues (Sarpal, 2002).

Kinesin-II is required for axonal transport of choline acetyltransferase in Drosophila

KLP64D and KLP68D are members of the kinesin-II family of proteins in Drosophila. Immunostaining for KLP68D and ribonucleic acid in situ hybridization for KLP64D demonstrated their preferential expression in cholinergic neurons. KLP68D was also found to accumulate in cholinergic neurons in axonal obstructions caused by the loss of kinesin light chain. Mutations in the KLP64D gene cause uncoordinated sluggish movement and death, and reduce transport of choline acetyltransferase from cell bodies to the synapse. The inviability of KLP64D mutations can be rescued by expression of mammalian KIF3A. Together, these data suggest that kinesin-II is required for the axonal transport of a soluble enzyme, choline acetyltransferase, in a specific subset of neurons in Drosophila. Furthermore, the data lead to the conclusion that the cargo transport requirements of different classes of neurons may lead to upregulation of specific pathways of axonal transport (Ray, 1999).

Characterization of the KLP68D kinesin-like protein in Drosophila: possible roles in axonal transport

KLP68D, a new kinesin-like motor protein in the fly has a domain that shares significant sequence identity with the entire 340-amino acid kinesin heavy chain motor domain. Sequences extending beyond the motor domain predict a region of alpha-helical coiled-coil followed by a globular 'tail' region; there is significant sequence similarity between the alpha-helical coiled-coil region of the KLP68D protein and similar regions of the KIF3 protein of mouse and the KRP85 protein of sea urchin. This finding suggests that all three proteins may be members of the same family, and that they all perform related functions. KLP68D protein produced in Escherichia coli is, like kinesin itself, a plus-end directed microtubule motor. In situ hybridization analysis of KLP68D RNA in Drosophila embryos indicates that the KLP68D gene is expressed primarily in the central nervous system and in a subset of the peripheral nervous system during embryogenesis. Thus, KLP68D may be used for anterograde axonal transport and could conceivably move cargoes in fly neurons different from those moved by kinesin heavy chain or other plus-end directed motors (Pasavento, 1994).

Functions of Kinesin-2 orthologs in other species

The spatiotemporal construction of the axon initial segment via KIF3/KAP3/TRIM46 transport under MARK2 signaling

The axon initial segment (AIS) is a compartment that serves as a molecular barrier to achieve axon-dendrite differentiation. Distribution of specific proteins during early neuronal development has been proposed to be critical for AIS construction. However, it remains unknown how these proteins are specifically targeted to the proximal axon within this limited time period. This study revealed spatiotemporal regulation in mice driven by the microtubule (MT)-based motor KIF3A/B/KAP3 (see Drosophila Klp64D) that transports TRIM46 (see Drosophila Trim9), influenced by a specific MARK2 (Drosophila homolog: Par-1) phosphorylation cascade. In the proximal part of the future axon under low MARK2 activity, the KIF3/KAP3 motor recognizes TRIM46 as cargo and transports it to the future AIS. In contrast, in the somatodendritic area under high MARK2 activity, KAP3 phosphorylated at serine 60 by MARK2 cannot bind with TRIM46 and be transported. This spatiotemporal regulation between KIF3/KAP3 and TRIM46 under specific MARK2 activity underlies the specific transport needed for axonal differentiation (Ichinose, 2019).

A neuron is a differentiated and polarized cell that consists of a cell body, several dendrites, and a long axon. To achieve the functional polarized morphology, immature neurons undergo spatiotemporal differentiation processes crucial for axon specification: outgrowth of the future axon followed by the construction of the axon initial segment (AIS), with its specific components to facilitate and maintain axonal differentiation (Ichinose, 2019).

Microtubules (MTs) are extended polymers composed of ~13 protofilaments that consist of alphaand beta-tubulin dimers whose asymmetric structure produces the polarity of MTs with a fast-growing/shrinking plus end and a less dynamic minus end. The polarity of MTs contributes to polarized intracellular trafficking as rails of motor proteins, such as kinesin super family proteins (KIFs) and dynein. KIF3, a member of the kinesin-2 family, forms a heterotrimeric complex that consists of KIF3A, KIF3B, and its cargo-binding adaptor, kinesin-associated protein 3 (KAP3) encoded by kifap3. KIF3/KAP3 is also reported to contribute to MT organization as well as cargo transport. Thus, KIF3/KAP3 has a fundamental property to produce the polarized morphology of the cells as well as the specific distributions of organelles and proteins via elaborate coordination of related molecules, including MTs (Ichinose, 2019).

Previously, the molecular interplay of cytoskeletal proteins has been proposed to be a critical intrinsic factor to generate axonal polarity in developing neurons. In developing axons, the specific proteins are transported into the future AIS and organized to construct the unique compartment, in which MTs are oriented uniformly with the plus ends toward the distal axon and the minus ends toward the cell body (plus-end out MTs). In contrast, dendritic MTs, especially in the proximal region, display a mixed-polarity orientation (mixed-polarity MTs). To date, various models and molecules have been proposed to participate in the construction of the specific MT orientation in neurons. TRIM46 belongs to the class I members of the tripartite motif (TRIM) family. It comprises an N-terminal RING finger, which is a general feature of E3 ubiquitin ligases, a B-box, a coiled-coil, a C-terminal subgroup one signature (COS) box, and a C-terminal FN3 and B30.2-like domain. The TRIM family generally consists of large protein complexes that possess ubiquitin-protein isopeptide ligase activity. In the neuron, TRIM46 is reported to have unique MT crosslinking activity localized in the most proximal part of the axon, the AIS. TRIM46 contributes to axon specification and the establishment of neuronal polarity by building closely spaced, parallel, stabilized MT fascicles in a uniformly 'plus-end out' orientation. This specific MT orientation and stability is thought to be a directional cue for the polarized trafficking of cargoes by motor proteins into the axon. However, it has been unclear how the spatiotemporal distribution of TRIM46 is produced under the elaborate coordination of specific molecules during early neuronal developmental stages. This study investigated the spatiotemporal regulation of TRIM46 accumulation at the AIS driven by the MT-based motor KIF3A/B/KAP3 under the specific MARK2 phosphorylation cascade (Ichinose, 2019).

This study reports one of the spatiotemporal mechanisms underlying AIS construction, which is a critical process for axon establishment and neuronal development. At the proximal part of the future axon, in which a low amount of MARK2 is localized, nonphosphorylated KAP3 loads TRIM46 and transports it via KIF3/KAP3 along MTs to the limited region of the future AIS. TRIM46 facilitates plus-end-out MT orientation and axon specification. In contrast, in the dendrite, in which the amount of MARK2 is highly enriched, TRIM46 does not accumulate because KAP3 phosphorylated via MARK2 cannot bind with and transport TRIM46. This study has revealed that phosphorylation via MARK2 alters the protein-protein interaction between KIF3/KAP3 and TRIM46, which directly facilitates the formation of cell polarity (Ichinose, 2019).

Intracellular transport of cargoes generally requires specific regulation for 'cargo loading' and 'cargo unloading.' As shown in this study, KIF3/KAP3 can 'load' TRIM46 as its specific cargo bound for the future AIS via the transient nonphosphorylated form of KAP3 only at approximately day in vitro (DIV) 4 (DIV4). Interestingly, this strict regulation via phosphorylation might not affect the direction and localization of KIF3 itself because both the KAP3 S60A and S60E mutants localized in axons. The regulated phosphorylation would instead affect the cargo loading activity of KIF3/KAP3. For spatiotemporal regulation, MARK2 localized in the somatodendritic region at DIV4 and later. This specific localization is also explained by previous studies showing that KIF3 initially transports the PAR-3/PAR-6/aPKC complex to the future axon, which suppresses MARK2 activity at the future axon during early developmental stages. Downregulation of MARK2 activity would facilitate the loading of TRIM46 by KAP3 and induce transport to the proximal part of the future axon to build up the AIS. This regulation of the KIF3/KAP3/TRIM46 complex would form the positive feedback loop of loading and transport of specific cargoes to further accelerate axon differentiation. However, it remains unknown how KIF3/KAP3 can 'unload' TRIM46 at the AIS. From the global localization of the KIF3/KAP3/TRIM46 complex on MTs in COS-7 cells, it is speculated that KIF3/KAP3/TRIM46 itself does not distinguish the specific structural destination cues. Instead, other regulators would be required to 'unload' TRIM46 from KIF3/KAP3 outside of the AIS to accumulate TRIM46 at only the AIS. The most likely candidate is the phosphorylation of TRIM46 by CDK5. Results from an in vitro kinase assay against TRIM46 indicated that the N-terminal region of TRIM46 containing the RING domain is phosphorylated by CDK5. S106 was identified in the RING domain as a phosphorylation site by CDK5. S106 is specific to the TRIM46 subfamily and is not conserved in the other TRIM subfamilies, such as TRIM36. Interestingly, this RING domain is reported to be necessary for TRIM46 accumulation at the AIS. In addition, CDK5 is also reported to be a key regulator of the AIS. More evidence is required to elucidate the precise unloading mechanism of TRIM46 under the CDK5 cascade (Ichinose, 2019).

Interestingly, this study found that both KIF3A and KAP3 were reduced in the kif3b+/− neurons. There should be a balance between KIF3A/B and KAP3 maintained at steady state, of which the ratio was previously reported to be 1:1:0.7, and the excess amount might be proteolyzed in the kif3b+/− neurons. Indeed, this stuy also overexpressed recombinant tagged KAP3 in neuronal cells, but only low expression of KAP3 was observed. Therefore, the strict balanced expression would explain the low coefficiency between tagged KAP3 and TRIM46 compared to that between endogenous KAP3 and TRIM46. However, tagged KAP3 was observed much more clearly in the kif3b+/− neuron than in the kif3b+/+ neuron because the expression level of KIF3 was potentially decreased to half, and there might be extra room for overexpressed KIF3 in KIF3/KAP3 balance. It might be interesting to investigate how KIF3 heterotrimers maintain their molecular balance in the future (Ichinose, 2019).

Depletion of KAP3 and KIF5 affected the distribution of AIS components. KIF5 has been reported to transport AnkG, which anchors βIV spectrin and other membrane proteins, such as Voltage-gated sodium channel (NaV) and L1-CAM, at the AIS. Because accumulated AnkG maintains axon/dendrite differentiation, KIF5 may facilitate axon differentiation by transporting AnkG. In fact, the data showing that depletion of KIF5 reduced Nav accumulation at the AIS are consistent with these previous studies. However, the detailed mechanism by which KIF5 transports AnkG to the AIS is still unknown, although it has been previously shown that KIF5 prefers stabilized MTs enriched in the AIS. More detailed mechanisms between KIF5 and AnkG as well as the regulation between KIF5 and TRIM46-stabilized MTs at the AIS should be investigated to solve this problem. However, KAP3 depletion also causes the reduction of Nav accumulation at the AIS. Moreover, it also causes Nav accumulation at the distal axon and growth cone in severe cases, which phenocopies depletion of spectrin. Considering that KIF3 transports αII spectrin, our results are consistent with previous results. Because αII spectrin is able to dimerize with either βIV spectrin at the AIS or βII spectrin at the distal axon, KIF3 may facilitate not only AIS construction but also whole axon differentiation by transporting its cytoskeletal and membrane proteins, such as axonal voltage-gated potassium channels. However, another study suggests that TRIM46 affects AIS construction by coordinating with AnkG. Depletion of AnkG reduced TRIM46 accumulation at the AIS and vice versa. The detailed mechanism between TRIM46, AnkG, KIF3, and KIF5 should be clarified to reveal the crosstalk between these molecules for the construction of the AIS. For example, it would be interesting to test whether KAP3 transports TRIM46 to the AIS in AnkG-depleted neurons. Considering that KIF5 might prefer TRIM46-stabilized MTs, there would be positive feedback assembly of the AIS by cooperative transport between KIF5 and KIF3. This molecular coordination would set up a selective barrier for the polarized trafficking of vesicles and organelles into the axon; axon/dendrite differentiation is further accelerated via previously reported mechanisms. Another major MT motor, KIF1A, does not seem to regulate axonal differentiation because the localization of Nav at the AIS was not affected by KIF1A depletion. However, the length of the AIS visualized with Nav immunostaining was slightly increased. Increased AIS length was also observed in auditory-deprived chick neurons. Considering that KIF1A transports synaptic vesicle precursors, it is possible that reduced presynaptic activity by KIF1A depletion increased the AIS length (Ichinose, 2019).

As shown in this study, the spatiotemporal molecular interplay of KIF3/KAP3/TRIM46 under MARK2 phosphorylation facilitates axon specification. AIS construction is important not only for selective transport but also for initiation of action potentials. Thus, manipulating neuronal excitability in this region would be a potential target for the treatment of epilepsy and psychiatric disorders. To reveal the relation between the regulation of AIS construction and neuronal diseases, further precise analyses, including both electrophysiological approaches in vivo and structural in vitro reconstruction studies, will contribute to a deeper understanding (Ichinose, 2019).

Reconstitution reveals motor activation for intraflagellar transport

The human body represents a notable example of ciliary diversification. Extending from the surface of most cells, cilia accomplish a diverse set of tasks. Predictably, mutations in ciliary genes cause a wide range of human diseases such as male infertility and blindness. In Caenorhabditis elegans sensory cilia, this functional diversity appears to be traceable to the differential regulation of the kinesin-2-powered intraflagellar-transport (IFT) machinery. This study reconstituted the first functional multi-component IFT complex that is deployed in the sensory cilia of C. elegans. The bottom-up approach revealed the molecular basis of specific motor recruitment to the IFT trains. The key component was identified that incorporates homodimeric kinesin-2 into its physiologically relevant context, which in turn allosterically activates the motor for efficient transport. These results will enable the molecular delineation of IFT regulation, which has eluded understanding since its discovery more than two decades ago (Mohamed, 2018).

The kinases male germ cell-associated kinase and cell cycle-related kinase regulate kinesin-2 motility in Caenorhabditis elegans neuronal cilia

Kinesin-2 motors power anterograde intraflagellar transport (IFT), a highly ordered process that assembles and maintains cilia. However, it remains elusive how kinesin-2 motors are regulated in vivo. A forward genetic screens was performed to isolate suppressors that rescue the ciliary defects of OSM-3-kinesin (homolog of mammalian homodimeric kinesin-2 KIF17) mutants in Caenorhabditis elegans. The C. elegans dyf-5 and dyf-18, which encode the homologs of mammalian male germ cell-associated kinase and cell cycle-related kinase, respectively, were identified. Using time-lapse fluorescence microscopy, it was shown that DYF-5 and DYF-18 are IFT cargo molecules and are enriched at the distal segments of sensory cilia. Mutations of dyf-5 and dyf-18 generate elongated cilia and ectopic localization of the heterotrimeric kinesin-2 (kinesin-II) at the ciliary distal segments. Genetic analyses reveal that dyf-5 and dyf-18 are important for stabilizing the interaction between IFT particles and OSM-3-kinesin. These data suggest that DYF-5 and DYF-18 act in the same pathway to promote handover between kinesin-II and OSM-3 in sensory cilia (Yi, 2018).

Kinesin-2 motors adapt their stepping behavior for processive transport on axonemes and microtubules

Two structurally distinct filamentous tracks, namely singlet microtubules in the cytoplasm and axonemes in the cilium, serve as railroads for long-range transport processes in vivo. In all organisms studied so far, the kinesin-2 family is essential for long-range transport on axonemes. Intriguingly, in higher eukaryotes, kinesin-2 has been adapted to work on microtubules in the cytoplasm as well. This study shows that heterodimeric kinesin-2 motors distinguish between axonemes and microtubules. Unlike canonical kinesin-1, kinesin-2 takes directional, off-axis steps on microtubules, but it resumes a straight path when walking on the axonemes. The inherent ability of kinesin-2 to side-track on the microtubule lattice restricts the motor to one side of the doublet microtubule in axonemes. The mechanistic features revealed in this study provide a molecular explanation for the previously observed partitioning of oppositely moving intraflagellar transport trains to the A- and B-tubules of the same doublet microtubule. These results offer first mechanistic insights into why nature may have co-evolved the heterodimeric kinesin-2 with the ciliary machinery to work on the specialized axonemal surface for two-way traffic (Stepp, 2017).

The cytoplasmic tail of rhodopsin triggers rapid rod degeneration in kinesin-2 mutants

Photoreceptor degeneration can lead to blindness and represents the most common form of neural degenerative disease worldwide. Although many genes involved in photoreceptor degeneration have been identified, the underlying mechanisms remain to be elucidated. This study examined photoreceptor development in zebrafish kif3a and kif3b mutants, which affect two subunits of the kinesin-2 complex. In both mutants, rods degenerated quickly, whereas cones underwent a slow degeneration process. Notably, the photoreceptor defects were considerably more severe in kif3a mutants than in kif3b mutants. In the cone photoreceptors of kif3a mutants, opsin proteins accumulated in the apical region and formed abnormal membrane structures. In contrast, rhodopsins were enriched in the rod cell body membrane and represented the primary reason for rapid rod degeneration in these mutants. Moreover, removal of the cytoplasmic tail of rhodopsin to reduce its function, but not decreasing rhodopsin expression levels, prevented rod degeneration in both kif3a and kif3b mutants. Of note, overexpression of full-length rhodopsin or its cytoplasmic tail domain, but not of rhodopsin lacking the cytoplasmic tail, exacerbated rod degeneration in kif3a mutants, implying an important role of the cytoplasmic tail in rod degeneration. Finally, this study showed that the cytoplasmic tail of rhodopsin might trigger rod degeneration through activating the downstream calcium signaling pathway, as drug treatment with inhibitors of intracellular calcium release prevented rod degeneration in kif3amutants. These results demonstrate a previously unknown function of the rhodopsin cytoplasmic domain during opsin-triggered photoreceptor degeneration and may open up new avenues for managing this disease (Feng, 2017).


Search PubMed for articles about Drosophila Klp64D or Klp68D

Avidor-Reiss, T., Maer, A. M., Koundakjian, E., Polyanovsky, A., Keil, T., Subramaniam, S. and Zuker, C. S. (2004). Decoding cilia function: defining specialized genes required for compartmentalized cilia biogenesis. Cell 117(4): 527-39. 15137945

Baqri, R., Charan, R., Schimmelpfeng, K., Chavan, S. and Ray, K. (2006). Kinesin-2 differentially regulates the anterograde axonal transports of acetylcholinesterase and choline acetyltransferase in Drosophila. J Neurobiol 66(4): 378-392. PubMed ID: 16408306

Dey, S., Banker, G. and Ray, K. (2017). Anterograde transport of Rab4-associated vesicles regulates synapse organization in Drosophila. Cell Rep 18(10): 2452-2463. PubMed ID: 28273459

Dey, S. and Ray, K. (2018). Cholinergic activity is essential for maintaining the anterograde transport of Choline Acetyltransferase in Drosophila. Sci Rep 8(1): 8028. PubMed ID: 29795337

Feng, D., Chen, Z., Yang, K., Miao, S., Xu, B., Kang, Y., Xie, H. and Zhao, C. (2017). The cytoplasmic tail of rhodopsin triggers rapid rod degeneration in kinesin-2 mutants. J Biol Chem 292(42): 17375-17386. PubMed ID: 28855254

Girotra, M., Srivastava, S., Kulkarni, A., Barbora, A., Bobra, K., Ghosal, D., Devan, P., Aher, A., Jain, A., Panda, D. and Ray, K. (2017). The C-terminal tails of heterotrimeric kinesin-2 motor subunits directly bind to alpha-tubulin1: Possible implications for cilia-specific tubulin entry. Traffic 18(2): 123-133. PubMed ID: 27976831

Greenspan, R. J. (1980). Mutations of choline acetyltransferase and associated neural defects. J. Comp. Physiol. 137: 83-92

Ichinose, S., Ogawa, T., Jiang, X. and Hirokawa, N. (2019). The spatiotemporal construction of the axon initial segment via KIF3/KAP3/TRIM46 transport under MARK2 signaling. Cell Rep 28(9): 2413-2426. PubMed ID: 31461655

Kulkarni, A., Khan, Y. and Ray, K. (2017). Heterotrimeric kinesin-2, together with kinesin-1, steers vesicular acetylcholinesterase movements toward the synapse. FASEB J 31(3): 965-974. PubMed ID: 27920150

Ligon, L. A., Tokito, M., Finklestein, J. M., Grossman, F. E. and Holzbaur, E. L. (2004). A direct interaction between cytoplasmic dynein and kinesin I may coordinate motor activity. J Biol Chem 279(18): 19201-19208. PubMed ID: 14985359

Matsuo, A., Bellier, J. P., Nishimura, M., Yasuhara, O., Saito, N. and Kimura, H. (2011). Nuclear choline acetyltransferase activates transcription of a high-affinity choline transporter. J Biol Chem 286(7): 5836-5845. PubMed ID: 21163949

Mattie, F. J., Stackpole, M. M., Stone, M. C., Clippard, J. R., Rudnick, D. A., Qiu, Y., Tao, J., Allender, D. L., Parmar, M. and Rolls, M. M. (2010). Directed microtubule growth, +TIPs, and kinesin-2 are required for uniform microtubule polarity in dendrites. Curr Biol 20(24): 2169-2177. PubMed ID: 21145742

Mohamed, M. A. A., Stepp, W. L. and Okten, Z. (2018). Reconstitution reveals motor activation for intraflagellar transport. Nature 557(7705): 387-391. PubMed ID: 29743676

Mukhopadhyay, B., Nam, S. C. and Choi, K. W. (2010). Kinesin II is required for cell survival and adherens junction positioning in Drosophila photoreceptors. Genesis 48(9): 522-530. PubMed ID: 20506262

Park, S. M., Littleton, J. T., Park, H. R. and Lee, J. H. (2016). Drosophila homolog of human KIF22 at the autism-linked 16p11.2 loci influences synaptic connectivity at larval neuromuscular junctions. Exp Neurobiol 25: 33-39. PubMed ID: 26924931

Pesavento, P. A., Stewart, R. J. and Goldstein, L. S. (1994). Characterization of the KLP68D kinesin-like protein in Drosophila: possible roles in axonal transport. J. Cell Biol. 127(4): 1041-8. PubMed ID: 7525600

Pflanz, R., Peter, A., Schafer, U. and Jackle, H. (2004). Follicle separation during Drosophila oogenesis requires the activity of the Kinesin II-associated polypeptide Kap in germline cells. EMBO Rep. 5(5): 510-4. 15088066

Ray, K., Perez, S. E., Yang, Z., Xu, J., Ritchings, B. W., Steller, H. and Goldstein, L. S. B. (1999). Kinesin-II is required for axonal transport of choline acetyltransferase in Drosophila. J. Cell Biol. 147: 507-517. 10545496

Sadananda, A., Hamid, R., Doodhi, H., Ghosal, D., Girotra, M., Jana, S. C. and Ray, K. (2012). Interaction with a kinesin-2 tail propels choline acetyltransferase flow towards synapse. Traffic 13(7): 979-991. PubMed ID: 22486887

Sarpal, R. and Ray, K. (2002). Dynamic expression of kinesin accessory protein in Drosophila. J. Biosci. 27: 479-487. 12381871

Sarpal, R., Todi, S. V., Sivan-Loukianova, E., Shirolikar, S., Subramanian, N., Raff, E. C., Erickson, J. W., Ray, K. and Eberl, D. F. (2003). Drosophila KAP interacts with the kinesin II motor subunit KLP64D to assemble chordotonal sensory cilia, but not sperm tails. Curr, Biol. 13(19): 1687-96. 14521834

Stepp, W. L., Merck, G., Mueller-Planitz, F. and Okten, Z. (2017). Kinesin-2 motors adapt their stepping behavior for processive transport on axonemes and microtubules. EMBO Rep 18(11): 1947-1956. PubMed ID: 28887322

Twelvetrees, A. E., Pernigo, S., Sanger, A., Guedes-Dias, P., Schiavo, G., Steiner, R. A., Dodding, M. P. and Holzbaur, E. L. (2016). The dynamic localization of cytoplasmic dynein in neurons is driven by Kinesin-1. Neuron 90(5): 1000-1015. PubMed ID: 27210554

Vuong, L. T., Mukhopadhyay, B. and Choi, K. W. (2014). Kinesin-II recruits Armadillo and Dishevelled for Wingless signaling in Drosophila. Development 141: 3222-3232. PubMed ID: 25063455

Weiner, A. T., Lanz, M. C., Goetschius, D. J., Hancock, W. O. and Rolls, M. M. (2016). Kinesin-2 and Apc function at dendrite branch points to resolve microtubule collisions. Cytoskeleton (Hoboken) 73(1): 35-44. PubMed ID: 26785384

Yasuyama, K., Kitamoto, T. and Salvaterra, P. M. (1996). Differential regulation of choline acetyltransferase expression in adult Drosophila melanogaster brain. J Neurobiol 30(2): 205-218. PubMed ID: 8738750

Yi, P., Xie, C. and Ou, G. (2018). The kinases male germ cell-associated kinase and cell cycle-related kinase regulate kinesin-2 motility in Caenorhabditis elegans neuronal cilia. Traffic 19(7): 522-535. PubMed ID: 29655266

Biological Overview

date revised: 28 June 2018

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