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Short Stop provides an essential link between F-actin and microtubules during axon extension

Department of Cell Biology and Center for Molecular Neuroscience, C-2210 Medical Center North,Vanderbilt University Medical Center, Nashville, TN 37232-0295, USA

View larger version (23K): [in a new window]   Fig. 1. Structures of Shot isoforms. (A) The F-actin/microtubule crosslinking Shot isoforms required for axon extension. The Shot L(A) isoform contains an N-terminal actin-binding domain, a central domain of 4000 amino acids and a C-terminal domain that contains EF-hand Ca2+-binding motifs, the GAS2 motif and other conserved sequences. The central domain contains a plakin-like region and 22 spectrin repeats (Strumpf and Volk, 1998). Numbers indicate the start and end positions of the different domains in the amino acid sequence and the percent amino acid identity between Drosophila Shot L(A) and mouse ACF7 (Leung et al., 1999) in the N-terminal, central and C-terminal domains. The other Shot isoform that crosslinks F-actin and microtubules, Shot L(B), differs from Shot L(A) only in the N-terminal leader sequence. ABD, actin-binding domain; EF, EF-hand; GAS2, GAS2 homology motif. (B) The structures of the four Shot N-terminal domains (Lee et al., 2000). Numbers indicate the start and end positions of the different features in the amino acid sequence. Type A and type B N-terminal domains differ in their leader sequences (residues 1-143 and 1-32, respectively) and contain two calponin homology motifs (CH1 and CH2). The type C domain contains yet another leader sequence (residues 1-210) and only the CH2 motif. The type D domain starts in the central rod domain.

View larger version (52K): [in a new window]   Fig. 2. Shot type A and B N-terminal domains associate with F-actin. (A) NIH3T3 fibroblasts transfected with type A, B, or C Shot N-terminal domains fused to GFP. CH, calponin homology domain. F-actin (red) is visualized using Cy3-conjugated phalloidin. Type A- and B-GFP fusions (green) co-localize with F-actin in the cortical region (arrowhead) and stress fibers (arrows) of transfected cells, but type C-GFP fusions (green) do not. Yellow (see merge panels) indicates the overlap between the F-actin and GFP distributions. Images are 1 µm confocal sections. Scale bar: 10 µm. (B) The in vitro actin-binding properties of Shot N-terminal domains. GST (Glutathione S-Transferase) fusions (2 µM) with either the second calponin homology domain (CH2) or the two calponin homology domains (CH1 + CH2) were incubated with F-actin (19.2 µM) and the samples separated into supernatant (S) or pellet (P) fractions by 1 hour of centrifugation at 150,000 g. The proteins in each fraction were then electrophoretically separated. The positions in the gel of the (CH1 + CH2)-GST fusion (open arrowhead), the CH2-GST-fusion (black arrowhead) and actin (arrow) are indicated. In the absence of F-actin, (lanes 1, 2, 7, 8), little fusion protein pellets. Little CH2-GST fusion is associated with F-actin (lanes 4, 6); most remains in supernatant fractions (lanes 3, 5). As CH2-GST is not well resolved from actin, the samples in lanes 3-4 were transferred electrophoretically to Immobilon P membranes (Millipore) and probed with anti-GST (lanes 5-6) to determine how much CH2 is associated with F-actin. CH2-GST largely remains in the supernatant after F-actin is pelleted (compare lane 5 with lane 6). By contrast, most of the (CH1+CH2)-GST fusion pellets with F-actin under these conditions (compare lane 9 with lane 10). (C) Scatchard plot of the binding reaction between the (CH1+CH2)-GST fusion and F-actin. Binding of the (CH1+CH2)-GST fusion to F-actin (4 µM) was measured at fusion protein concentrations of 100, 200, 300 and 400 nM. The relative amounts of proteins in the supernatant and pellet were determined by quantitative densitometry of silver-stained SDS-polyacrylamide gels. Three data sets were averaged; individual values differed from the average by no more than 9%. Vertical axis, (CH1+CH2)–GST bound/free (CH1+CH2)–GST (µM–1); horizontal axis, bound (CH1+CH2)–GST/F-actin.

View larger version (65K): [in a new window]   Fig. 3. The C-terminal domain of the long Shot isoforms (C-Shot L) binds to microtubules. (A) C-Shot L contains two EF-hand motifs (hatched) (Ikura, 1996) and homology to GAS2 (black) (Brancolini et al., 1992). EGY48 yeast cells (Gyuris et al., 1993) grow on leucine-depleted selective medium when they express the C-Shot L-lexA fusion and an -tubulin-activation domain fusion, but do not grow when they express a bicoid-lexA fusion and an -tubulin-activation domain fusion. The cDNA inserts of the two clones encode the C-terminal regions of tubulin 1 (amino acids 344 to 450, Accession Number, P06603) and tubulin 3 (amino acids 343 to 450, Accession Number P06605) (Theurkauf et al., 1986). EGY48 yeast also do not grow on selective medium when they contain the C-Shot L-lexA fusion and the activation domain alone (data not shown). (B) Summary of the qualitative ability of C-Shot L fragments to associate with microtubules in transfected cells. ++, as in E,K; +/–, as in H. (C-E) An NIH3T3 cell transfected with a C-Shot L-GFP expression vector. (F-H) A cell transfected with a GFP fusion to sequences C-terminal to the GAS2 sequence. (I-K) Cells treated with 1 µM nocodazole for 20 minutes. All cells transfected with the GAS2 motif-GFP expression vector (arrows) still contain microtubules, whereas no control cells (arrowhead) contain detectable microtubules. (L-N) Cells transfected with a GFP fusion to an N-terminal fragment from isoform Shot L(C) that contains the second calponin homology repeat and the plakin-like domain. (C,F,I,L) Microtubule distribution. (D,G,J,M) GFP expression and distribution. (E,H,K,N) Overlap (yellow) between microtubule (red) and GFP (green) distributions. Only GFP fusions with C-Shot L fragments that contain the GAS2 motif associate strongly with microtubules (E,K) and stabilize microtubules in transfected cells against nocodazole treatment (I-K). GFP fusions with a C-terminal fragment lacking the GAS2 motif associate weakly (H, arrow), and GFP fusions with the Shot plakin-like domain do not associate with microtubules (N). Images in C-N are 1 µm confocal sections. Scale bars: 10 µm.

View larger version (103K): [in a new window]   Fig. 4. Long isoforms of Shot crosslink F-actin and microtubules and are found in growth cones and neuronal processes. (A-D) Structures (arrows) containing F-actin, Shot L(A)-GFP and microtubules found in transfected NIH 3T3 cells expressing Shot L(A)-GFP. (E-H) Shot L(C)-GFP is associated with microtubules (arrows). (I-L) Shot L(A)-GAS2-GFP no longer clearly associates with microtubules. Most Shot L(A)-GAS2-GFP is diffusely distributed and some appears to be associated with F-actin (yellow in L). (M-P) Shot L(A)-EF-GFP forms aggregates (arrows) containing F-actin, Shot and microtubules. (Q-T) The distributions of F-actin (Q), Shot long isoforms (R) and microtubules (S) in neurites in culture. Arrow indicates tip of an extending neurite. (T) Merge of Q-S. (A,E,I,M,Q) F-actin (red); (B,F,J,N) Shot GFP fusion protein (green); (C,G,K,O,S) Microtubules (white). In D,H,L,P,T, microtubules are shown in blue. Structures that contain F-actin, Shot-GFP and microtubules appear white, structures that contain only microtubules and Shot-GFP appear blue-green, and structures that contain only F-actin and Shot-GFP appear yellow. (U) Shot L(A)-GFP labels dendrites (den) and axon bundles (ISN, SNa) when expressed in neurons in a Drosophila embryo. The dendrites shown are those of the lateral chordotonal cluster. ISN and SNa axons are indicated. Similar levels of expression and localization patterns were observed for other Shot L(A)-GFP derivatives. All panels are 1 µm confocal sections, except U, which is a 5 µm confocal stack. Scale bars: 10 µm in A-P; 25 µm in Q-T.

View larger version (159K): [in a new window]   Fig. 5. The F-actin/microtubule crosslinking activity and Ca2+ binding domains of Shot L(A) are required in neurons for motor axon extension. (A-H) The pattern of muscle innervation in late stage 16 Drosophila embryos. (A) Wild type. In each abdominal hemisegment, the ISN motor axons innervate dorsal muscles, and form three characteristic neuromuscular junctions (1-3) in each abdominal hemisegment (short arrows). The dorsal trunk of the trachea (T) runs approximately between the dorsalmost two connections. The SNa contacts lateral muscles and bifurcates above muscle 12 into a dorsal (d) and a lateral (l) branch (concave arrows). The ISNb (long arrows) innervates ventral muscles 12, 13, and the cleft between muscles 6 and 7. (B) shot3 null mutant. The ISN stalls (s) at the approximate position where it would normally make the second dorsalmost muscle contact (short arrow). The SNa stalls (s) at the point where it would normally bifurcate (concave arrow). The ISNb stalls (s) in the ventral muscle field (long arrows), and fails to reach muscle 12. (C-H) The indicated transgenes are expressed specifically in all neurons in shot3 mutant embryos. (C) Shot L(A)-GFP expression restores normal muscle innervation. All Shot L(A)-GFP derivatives produced green fluorescence and were present in axons when expressed in Drosophila embryonic neurons (data not shown). (D) Shot L(C)-GFP expression does not rescue normal muscle innervation. (E) Shot L(A)-rod1-GFP (Table 1) expression restores normal muscle innervation. (F) Shot L(A)-GAS2-GFP expression does not rescue normal muscle innervation. (G) Shot L(C)-GFP and Shot L(A)-GAS2-GFP co-expression does not rescue normal muscle innervation. (H) Shot L(A)-EF-hand-GFP expression does not rescue normal muscle innervation. Scale bar: 15 µm. Anterior, left; dorsal, top.

View larger version (96K): [in a new window]   Fig. 6. The F-actin/microtubule crosslinking activity and Ca2+-binding domains of Shot L(A) are required in neurons for sensory axon extension. Sensory neurons and their axons in abdominal hemisegments a1-a7 in filleted stage 16 Drosophila embryos. (A) Wild-type. The PNS neurons in each hemisegment are organized into dorsal (d), lateral (l) and two ventral (v,v') clusters. Neurons in the dorsal and lateral clusters send their axons (arrows) in the intersegmental nerve (ISN) to CNS targets. (B) shot3 null mutant. Sensory axons are rudimentary or absent (arrows). Cell bodies remain at the appropriate positions. (C-G) The indicated transgenes are expressed in neurons in shot3 mutant embryos. (C) Shot L(A) expression restores sensory axons (arrows). (D) Shot L(C)-GFP expression fails to rescue sensory axon extension (arrows). (E) Shot L(A)-GAS2-GFP expression fails to rescue sensory axon extension (arrows). (F) Co-expression of Shot L(C)-GFP and Shot L(A)-GAS2-GFP fails to rescue sensory axon extension (arrows). (G) Shot L(A)-EF-hand-GFP expression fails to rescue sensory axon extension (arrows). Scale bar: 25 µm. Anterior, left; dorsal, top.

View larger version (24K): [in a new window]   Fig. 7. Domains in Shot L(A) required for axon extension. Neuronal expression of Shot L(A)-GFP rescues (+) motor and sensory axon extension defects in shot mutants. Shot L(A) contains an N-terminal actin-binding domain (ABD, black box), a central rod domain consisting of a plakin-like region (gray box), 22 spectrin repeats (hatched box), and a C-terminal domain with Ca2+- (EF-hand) and microtubule- (GAS2) binding motifs. Shot L(C)-GFP contains no ABD and has no rescue activity (–). Derivatives of Shot L(A)-GFP that lack the EF-hand, GAS2 homology or the spectrin repeat sequences were tested for motor and sensory axon extension activity. Shot L(C) and Shot L(A)-GAS2 also have no rescue activity when co-expressed.