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Vol. 16, Issue 9, 4225-4230, September 2005
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* Department of Biology, Indiana University, Bloomington, IN 47405;
McArdle Laboratory, University of Wisconsin, Madison, WI 53706
Submitted February 10, 2005;
Revised June 13, 2005;
Accepted June 15, 2005
Monitoring Editor: John Pringle
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Hernandez et al., 2004). Recent work suggests that Abl also influences microtubule polymerization in the axon growth cone via interactions with Orbit and that a mouse Abl-related protein, Arg, can cross-link F-actin with microtubules in the cell periphery (Lee et al., 2004; Miller et al., 2004). In part, the influence of Abl on F-actin-dependent processes is mediated by its regulatory interaction with Ena/VASP proteins, which can modulate actin filament length, branching pattern, and bundle formation (reviewed by Krause et al., 2003; Kwiatkowski et al., 2003). Ena/VASP proteins have three conserved regions. The N-terminal EVH1 domain (133 amino acids, Gertler et al., 1996) can bind the focal-adhesion proteins vinculin and zyxin, as well as the growth-cone guidance receptor Robo/Sax3. The central proline-rich region can bind profilin, which facilitates the addition of G-actin monomers to F-actin plusends. It can also bind Abl and other SH3-domain proteins. The C-terminal EVH2 domain (226 amino acids, Gertler et al., 1996) has both G- and F-actin binding sites and has been shown to mediate Ena/VASP multimerization (reviewed by Krause et al., 2003; Kwiatkowski et al., 2003).
In Drosophila development, Ena and Abl are known to have an antagonistic relationship. Zygotic mutations in the ena or Abl gene cause axon growth-cone guidance defects and lethality, but normal axon guidance and viability can be restored by combining ena and Abl mutations (reviewed by Krause et al., 2003). Mutation of the Abl gene in the female germline eliminates maternal Abl protein from embryos, resulting in aberrant epithelial cell shape and defective dorsal closure. In embryonic epithelial cells that lack Abl, Ena mislocalization to the apical region causes overgrowth of F-actin based microvilli (Grevengoed et al., 2001, 2003). Overall, it is evident that Ena protein helps form long, unbranched actin filaments and that Abl negatively regulates Ena activity and/or localization. Whether Abl controls Ena by simply binding it, by tyrosine phosphorylation, or by some other means remains unclear (reviewed by Krause et al., 2003).
Kinesin-1 proteins are classic, plus-end-directed microtubule motors that function in mRNA localization, organelle movement, and axonal transport (reviewed by Vale, 2003). Kinesin-1s are not known to have direct roles in cell shape changes, axon guidance, or cell migration. Through protein-binding and genetic tests, we have identified physical and functional interactions between Drosophila kinesin-1, Abl, and Ena. Our results suggest that in addition to its influence on actin cytoskeleton organization, Abl signaling can influence microtubule-based anterograde fast transport in neurons by regulation of Ena, which binds the stalk-tail region of Khc. That region of Khc is thought to be important for kinesin-1 autoregulation and cargo interaction.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Chromosomal-deletion strains and all other fly strains, unless noted otherwise, were obtained from the Bloomington Stock Center (Indiana University, Bloomington, IN). Ena protein was overexpressed in flies by generating strains carrying a Gal4-UAS:ena transgene, P{UAS-ena, w+}1A1 (Comer et al., 1998), and one of the following drivers: 1) Act5C-GAL4 (P{w+mc = Ay-GAL4}25), 2) HSP70-GAL4 (P{w+mC = GAL4-Hsp70.PB}2), or 3) elav-GAL4 (P{w+mW.hs = GawB}elavC155).
To gain an unbiased view of the severity of larval posterior paralysis (tail flipping) as a function of genotype, 25–50 randomly selected wandering third-instar larvae from each test class were studied as described previously (Martin et al., 1999). Each cross was done and scored at least twice, and tests of Abl, ena, and Khc genetic interactions were done double-blind with negative and positive controls included. Wandering third-instar larvae were scored as tail flippers if at least two posterior segments curved up away from the substrate during the crawling cycle.
Yeast Two-hybrid and S2 Expression Constructs
A "bait" cDNA encoding the C-terminal 243 amino acids of Ena fused to the GAL4 DNA-binding domain was used to screen Drosophila larval cDNAs ("prey") fused to sequences encoding the GAL4 activation domain, as described previously (Ahern-Djamali et al., 1999). Blue colonies were isolated and retested, and the prey constructs in positive clones were subjected to sequence analysis. Full-length Ena, truncated Ena, and Khc transfection constructs were made by ligating cDNAs (Drosophila Genomics Resource Center, http://dgrc.cgb.indiana.edu/) into the pPacPL expression vector (Ahern-Djamali et al., 1998). Mutant ena cDNAs included one that truncated the C-terminal 52 codons (Ahern-Djamali et al., 1998), and another cut at a unique NcoI site that truncated the C-terminal 243 codons (this publication).
Immunoprecipitation
Drosophila S2 cells (1 x 107), either nontransfected or cotransfected (Gertler et al., 1995) with cDNAs that expressed Ena and Khc, were lysed for 30 min on ice in 1 ml of extraction buffer (Saxton et al., 1991). Adult fly heads were detached by vigorously shaking flies after freezing at -80°C or in liquid nitrogen. The heads and bodies were sorted at -20°C by passage through a no. 20 sieve, which retained bodies, and a no. 40 sieve, which retained heads. Frozen heads were disrupted with a cold ceramic mortar and pestle and then suspended in 1 ml of 4°C extraction buffer for every 0.12 g of starting material. Lysate or homogenate was clarified by centrifugation at 15,000 x g for 20 min and then at 30,000 x g for 30 min. The resulting S2 or head cytosol was immunoprecipitated, as described previously (Gertler et al., 1995). Briefly, 1 µg of anti-GFP (8367–1, Clonetech, Mountain View, CA), 1 µg of anti-Ena-carboxy-terminal (Gertler et al., 1989), 1 µg of anti-Drosophila-Khc (Cytoskeleton, Denver, CO), or 15 µl of Flyk2 anti-Khc (Saxton et al., 1991) was added to 300-µl fractions of cytosol (
400 heads) and rocked for 1 h at 4°C. Protein A- or G-coated beads (Santa Cruz Biotechnology, Santa Cruz, CA) were added, and the suspensions were rocked overnight at 4°C and then washed three times with 500 µl of extraction buffer and boiled in SDS-PAGE sample buffer. SDS-PAGE (7.5% gels), blotting, and staining were done as described previously (Saxton et al., 1988; Gertler et al., 1995), except that an ECL system was used for detection (Amersham, Piscataway, NJ). Primary antibodies used to detect Ena and Khc included rabbit anti-Khc R1.5 (Saxton et al., 1988) and rabbit anti-Ena-amino-terminal (Gertler et al., 1989), as well as the antibodies described above. Prestained molecular weight standards were included in the SDS-PAGE, and their positions were marked on blots before blocking and antibody incubations.
Immunolocalization and Microscopy
Larval dissection, fixation, immunostaining, and confocal fluorescence microscopy were done as described previously (Hurd and Saxton, 1996) except that before anti-Ena staining, formaldehyde-fixed larvae were postfixed in -20°C methanol for 10 min and then washed in phosphate-buffered saline with 0.01% Tween 20. Dissection, fixation, and staining of mutant and control larvae were done in the same dish, and then micrographs were generated with identical optical and electronic settings using a Bio-Rad MRC600 confocal microscope (Richmond, CA; Hurd and Saxton, 1996). The antibodies and dilutions used were rabbit anti-Drosophila-synaptotagmin at 1:500 (Littleton et al., 1993), mouse anti-Drosophila-cysteine-string-protein at 1:250 (Zinsmaier et al., 1994), and rabbit anti-Ena-amino-terminal or rabbit anti-Ena-carboxy-terminal at 1:100 (Gertler et al., 1989). The secondary antibodies (at 1:500) were goat anti-rabbit-IgG:FITC (Jackson ImmunoResearch Laboratories, West Grove, PA), goat anti-rabbit-IgG:ALEXA-488, and goat anti-mouse-IgG:ALEXA-594 (both from Molecular Probes, Eugene, OR). Images were manipulated using NIH Image and Adobe Photoshop 7.0 (San Jose, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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, 1999), an RNA binding protein (lark), a putative laminin receptor/ribosomal S2 protein (stubarista), and the C-terminal 261 amino acids of Khc (Figure 1B). That portion of Khc includes the alpha helical "coil-2" and globular "tail" regions that bind kinesin light chain (Klc), that mediate cargo interaction, and that can inhibit ATPase activity of the N-terminal motor domain (reviewed by Verhey and Rapoport, 2001). Given previous work on Ena/VASP showing that it modulates actin cytoskeleton organization, the Khc interaction was particularly interesting. To determine if Khc and Ena bind one another in Drosophila cytosol, we tested for coimmunoprecipitation (Figure 2). First, cytosol was prepared from Drosophila S2 cells that had or had not been cotransfected with full-length Ena and Khc expression constructs. In nontransfected cells, anti-Ena antibodies precipitated Khc but anti-Khc antibodies did not precipitate detectable Ena, perhaps because of weaker antibody-antigen interaction. However, in overexpressing cells, each antibody clearly coprecipitated detectable amounts of both proteins (Figure 2A; homotypic staining is not published). To determine if Ena-Khc binding in cytosol was mediated by the region of Ena used in the yeast two-hybrid test, additional immunoprecipitations were done with S2 cells that expressed C-terminal truncations of Ena (Figure 2B). Expression of truncated Khc stalk/tail constructs was unsuccessful, so the reciprocal tests were not done. The coprecipitation results with truncated Ena indicated that amino acids 441–632 of Ena were required for Ena-Khc binding in cytosol. This segment includes parts of the proline-rich and EVH2 regions that contain known binding sites for Abl, profilin, actin, and Ena (see Introduction).
To determine if Ena and Khc bind one another in cytosol from intact neuronal tissue, immunoprecipitations were also performed with adult fly heads (Figure 2C). When anti-Khc antibodies were used, Ena coprecipitation was sometimes weak and sometimes not detected. However, anti-Ena antibodies consistently precipitated Khc. Considered together with the observations with extracts from cultured cells, the coprecipitation from adult heads shows that Ena, a protein previously known for binding the actin cytoskeleton and influencing its organization, can also bind the microtubule motor protein kinesin-1 in Drosophila. This interaction and the fact that Khc, Abl, and Ena are neuronal proteins raised two questions: does Ena-Khc binding facilitate the anterograde transport of Ena into the axon, and/or does Ena have a kinesin-1 regulatory function?
Ena Does Not Behave Like a Fast-Axonal-Transport Cargo
To test the possibility that Ena is a fast-transport cargo of kinesin-1, we studied the effects of Khc mutations on Ena distribution in segmental nerves by immunostaining and fluorescence microscopy. Known fast-axonal-transport cargoes, such as vesicle-associated synaptic terminal proteins and mitochondria, accumulate in axonal swellings caused by mutation of kinesin-1 or dynein subunits (Hurd and Saxton, 1996; Gindhart et al., 1998; Bowman et al., 1999; Martin et al., 1999). Slow-transport materials, such as tubulin, appear evenly distributed in the nerves of such motor mutants and do not accumulate in axonal swellings (Hurd and Saxton, 1996). Ena staining patterns in segmental nerves were compared with those of cysteine string protein (CSP), a known fast-transport component, in wild-type and partial-loss-of-function (hypomorphic) Khc mutant larvae (Figure 3). In wild-type larvae, CSP staining was faintly punctate in segmental nerves and strong in the boutons of motor-axon terminals (Figure 3, A and C). Anti-Ena staining showed a different pattern: nonpunctate longitudinal streaks in the axon-rich cores of nerves and diffuse localization throughout terminals (Figure 3, A and C). In Khc mutants, CSP accumulated to high levels in axonal swellings, consistent with its being a fast-axonal-transport cargo (Figure 3B). Ena did not accumulate in those swellings but instead maintained an even axonal distribution (Figure 3B). An obvious reduction in staining for both proteins in axon terminals (Figure 3D) was probably due to the substantial reduction in terminal size caused by Khc mutations (Hurd and Saxton, 1996). The lack of Ena accumulation in axonal swellings suggests that it is not carried by kinesin-1 as a fast-axonal-transport cargo. However, intermittent Ena-Khc interactions could facilitate Ena anterograde movement by slow axonal transport (Brown, 2003).
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). Of 137 deletions that collectively uncovered 75% of the Drosophila genome, 11 had EK activity (see Table S1 in Supplementary Materials). One of those deletions, Df(3L)81k19, removed a portion of the left arm of chromosome 3. To map the EK locus within that area, several smaller deletions were tested. Five with breakpoints in the 73A-74F polytene region had EK activity (Figure 4). The common deleted interval uncovered Baldspot, Abl, and three other predicted genes.
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) increased tail-flipping penetrance to 50%, which was comparable to that seen in similar EK tests of a severe Klc mutation (Martin et al., 1999). These results suggest that wild-type Abl and its interacting proteins Dab and/or Nrt have overlapping positive influences on the axonal transport activity of Drosophila kinesin-1.
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). In a third test, we studied the effect of ena mutations on the synthetic Abl-Khc phenotypes. As opposed to the tail-flipping and abundant swellings seen in Khc16/Khc+; Abl1/Abl+ double heterozygotes, triple heterozygotes (Khc16 ena+/Khc+ ena210; Abl1/Abl+) showed no tail flipping and a marked reduction in axonal swellings (Figure 5). Thus, in a background sensitized by reducing the dose of Khc by one-half, axonal transport problems caused by reduced Abl activity can be prevented by also reducing Ena activity. This suggests that endogenous Ena, when not inhibited by Abl, has a negative influence on kinesin-1-driven axonal-transport.
Ena/VASP, when freed of negative regulation by Abl, facilitates the growth of abnormally long, unbranched actin filaments (Bear et al., 2002). If the Abl-Khc synthetic interaction phenotypes reflect nonspecific changes in the cytoplasm, such as increased viscosity due to F-actin overgrowth, one would expect similar synthetic interactions between Abl and other axonal transport mutations. To test this, we looked for interactions between Abl and Dhc64C, which encodes the force-producing subunit of cytoplasmic dynein, a retrograde axonal transport motor. Homozygous hypomorphic Dhc64C mutations cause axonal-swelling and tail-flipping phenotypes similar to those caused by Khc mutations (Martin et al., 1999). Larvae doubly heterozygous for an Abl null and for Dhc64Cek1, a null-like allele (Martin et al., 1999), showed neither tail flipping nor axonal swelling enhancement. Thus, reduced Abl dosage does not cause a general hindrance of all fast-axonal-transport. This suggests that the Ena-Khc physical interaction and the Abl-ena-Khc genetic relationships reflect specific functional interactions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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).
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). Thus, Ena could inhibit transport by destabilizing Khc dimerization, by enhancing tail/motor domain interaction, or by disturbing Khc-Klc interactions. The C-terminal portion of Ena that binds Khc mediates Ena multimerization and binding of Abl, profilin, and other proteins (reviewed by Korey and Van Vactor, 2000; Krause et al., 2003; Kwiatkowski et al., 2003). One possibility raised by the three-way Khc-Abl-ena genetic interaction is that binding of activated Abl to Ena causes a conformational change that inhibits Ena binding to Khc. Tests of the effects of Abl overexpression on Ena-Khc interaction in cultured cells showed no consistent effect (Ahern-Djamali and Martin, unpublished results). However, as with tests of Ena overexpression (this report and Comer et al., 1998), the activation and control of Abl and Ena may be too complex for this sort of approach to be informative.
The results of this study add to evidence that Abl influences both F-actin and microtubule cytoskeleton activities. Abl control of F-actin architecture and lamellipodial activity, through regulation of Ena/VASP proteins, is well known (see Introduction). It also has been demonstrated recently that Abl can regulate microtubule plus-end-polymerization dynamics at the periphery of neuronal growth cones by helping transmit signals from Robo receptors to orbit, which is a microtubule plus-end-binding protein (Lee et al., 2004). Our results, which suggest that Abl regulates a plus-end-directed microtubule motor protein, support the idea that Abl acts as a signaling "node" to coordinate the responses of F-actin- and microtubule-based processes to extracellular signals.
Kinesin-1 has a variety of different cargoes that employ different linkage mechanisms (Verhey and Rapoport, 2001; Gunawardena and Goldstein, 2004). Thus, in addition to global kinesin-1 regulators, there may be specific regulators that influence the transport of specific subsets of cargoes. We suspect this is the case for Ena, because kinesin-1-driven anterograde transport of mitochondria appears normal in Khc Abl double-heterozygous mutant larval nerves (Bader, Pilling, and Saxton, unpublished results). Future studies of the transport of other axonal organelles should be informative. In addition to this issue of specificity, other key questions come to mind: Do transmembrane receptors like Robo use Abl and Ena to make axonal transport responsive to extracellular cues? Do Abl and Ena help coordinate changes in actin and growth cone dynamics with changes in fast-axonal-transport? Future experimentation that addresses questions about the Abl-Ena-Khc relationships should produce some interesting answers and may provide new insights into coordination between F-actin- and microtubule-based processes.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
These authors contributed equally to this work.
Address correspondence to: William Saxton (bsaxton{at}bio.indiana.edu).
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