In many eukaryotes, coordination of chromosome segregation with cell cleavage relies on the patterned interaction of specific microtubules with actin filaments through dedicated microtubule plus-end tracking proteins (+TIPs). We argue that these likely involve the neuronal Shot‐PH isoform, which is characterised by a large, unexplored central plakin repeat region (PRR) similarly existing also in mammalian spectraplakins. In addition to actin‐MT cross‐linkage, we find strong indications that Shot executes redundant MT bundle‐promoting roles that are F‐actin‐independent. Here we have gained new understanding by showing that the F‐actin interaction must be finely balanced: altering the properties of F‐actin networks or deleting/exchanging Shot's CH domains induces changes in Shot function ‐ with a Lifeact‐containing Shot variant causing remarkable remodelling of neuronal microtubules. The underlying mechanisms are best understood for Drosophila’s spectraplakin Short stop (Shot) and believed to involve cytoskeletal cross‐linkage: Shot's binding to microtubules and Eb1 via its C‐terminus has been thoroughly investigated, whereas its F‐actin interaction via N‐terminal calponin homology (CH) domains is little understood. Spectraplakin deficiency in mouse or Drosophila causes severe decay of microtubule bundles and reduced axon growth. Key regulators of neuronal microtubules are the spectraplakins, a well‐conserved family of cytoskeletal cross‐linkers that underlie neuropathies in mouse and humans. Understanding microtubule regulation is therefore an essential aspect of axon biology. The growth and maintenance of axons require loose microtubule bundles that extend through their entire length. Some of the combinatorial possibilities are diagrammed for variants of isoform A the other N-terminal sequences may exist in similar combinations.Īxons are the long and slender processes of neurons constituting the biological cables that wire the nervous system. The numbers refer to the amino acid residues at the junction of these different protein domains for each isoform depicted. A second C-terminal domain (long cross-hatched box) is associated with a shorter rod domain. Alternative splicing in the middle of the protein can cause the insertion of a 300 amino acid coiled-coil sequence (long white box), and alternative splicing of the C-terminal domain of the long isoform generates additional diversity (small black or white boxes). Isoform C contains a partial ABD (black box), following a unique sequence of 210 amino acids (vertically striped box), and isoform D contains no globular N-terminal domain. Two isoforms (A and B) contain complete predicted actin binding domains (black box labeled ABD), following unique sequences of 143 (horizontal striped box) or 32 amino acids (cross-hatched box), respectively. Four different N-terminal sequences were identified. B, Some of the predicted proteins encoded by the shot gene. The exons encoding the GAS2 domain are indicated. Asterisks denote the exons encoding the Ca 2-binding EF-hand motifs (Ikura, 1996). The exons indicated by brackets 1 and 2 encode the predicted actin binding domain found in isoforms A and B the exons indicated by bracket 2 are the only portion of the predicted actin binding sequences retained in the isoform C mRNA. Black box indicates an alternatively spliced exon in the sequence encoding the long isoform C-terminal domain cross-hatched boxes indicate the exons encoding the C-terminal domain of the shorter isoform. Gray boxes indicate exons common to longer isoforms white boxes indicate alternatively spliced exons.

An additional two exons that encode the 5 ends of isoforms A and B, respectively, are located upstream of this genomic region (Gregory and Brown, 1998), and their sizes and positions have not been analyzed in this study. The boxes represent exons (35) mapped in the shot gene by comparison of cDNA with genomic DNA sequence (Berkeley Drosophila Genome Project), and the patterns of alternative splicing are indicated. Inverted triangles indicate the sites of P-element insertions, and the arrows in the 5 end of the gene indicate the likely transcription start points for the mRNAs encoding isoforms C and D, as inferred from the corresponding cDNAs. A, The genomic structure of the shot/kak gene at 50C6-10 on chromosome II and P-element insertion sites. Multiple transcriptional starts and alternative splicing throughout the short stop/kakapo gene generate protein isoforms predicted to differ in their actin binding properties (isoform types A-D), rod domain length, and C-terminal domains.

Box shot 3d alternative