The Drosophila Gene abnormal spindle Encodes a Novel Microtubule-associated Protein That Associates with the Polar Regions of the Mitotic Spindle

Abstract

abnormal spindle, a gene required for normal spindle structure and function in Drosophila melanogaster, lies immediately adjacent the gene tolloid at 96A/B. It encodes a 220-kD polypeptide with a predicted pI of 10.8. The recessive mutant allele asp1 directs the synthesis of a COOH terminally truncated or internally deleted peptide of ∼124 kD. Wild-type Asp protein copurifies with microtubules and is not released by salt concentrations known to dissociate most other microtubule-associated proteins. The bacterially expressed NH2-terminal 512-amino acid peptide, which has a number of potential phosphorylation sites for p34cdc2 and MAP kinases, strongly binds to microtubules. The central 579-amino acid segment of the molecule contains one short motif homologous to sequences in a number of actin bundling proteins and a second motif present at the calmodulin binding sites of several proteins. Immunofluorescence studies show that the wild-type Asp protein is localized to the polar regions of the spindle immediately surrounding the centrosome. These findings are discussed in relation to the known spindle abnormalities in asp mutants.

Structure and microtubule-nucleation activity of isolated Drosophila embryo centrosomes characterized by whole mount scanning and transmission electron microscopy

“…The anti-alphatubulin DM1A and anti-c-tubulin GTU88 antibodies were obtained from Sigma (Munich, Germany). The anti-CNN antibody (Li and Kaufman 1996), and the anti-asp antibody (Saunders et al 1997) were kind gifts of Drs. Kaufman (Bloomington, USA) and Glover (Cambridge, UK), respectively.…”

The Centrosome and Its Duplication Cycle

“…The most commonly affected gene in microcephaly is aspm (abnormal spindle-like microcephaly associated, MCPH5) (Thornton and Woods 2009). In cultured human cells, ASPM localizes to centrosomes and spindle poles, similar to its fly and worm ortholog (Saunders et al 1997;Kouprina et al 2005;van der Voet et al 2009;Noatynska et al 2012). Neuroprogenitors depleted of ASPM fail to orient the mitotic spindle perpendicular to the ventricular surface of the neuroepithelium giving asymmetric, rather than symmetric, divisions (Fish et al 2006).…”

The meiotic spindle of theDrosophilaoocyte: the role of Centrosomin and the central aster

“…As the product of the gene abnormal spindle (asp) appears to perform a major role in the organization and dynamic of the central spindle MTs in both mitotic cells and spermatocytes, we asked if this protein was also present within the central region of the post-anaphase meiosis I female spindle. The MT minus-end binding protein Asp localizes to the polar regions of mitotic and meiotic spindles (Saunders et al, 1997) and exhibits a striking enrichment at the terminal regions of their mid-zone MTs (Wakefield et al, 2001;Riparbelli et al, 2002).…”

“…Mouse monoclonal anti-β-tubulin (Boehringer-Mannheim, UK) was used at a 1:200 dilution; rat monoclonal YL1/2 directed against tyrosinated α-tubulin (Harlan Sera-Lab, England) at a dilution of 1:20; a mouse anti-γ-tubulin monoclonal antibody (Sigma, St Louis, MO) at 1:100; a rabbit polyclonal anti-CP190 serum Rb188 (Whitfield et al, 1988) at 1:400 dilution; a rabbit polyclonal anti-Asp serum Rb3133 (Saunders et al, 1997) at 1:50 dilution; a rabbit polyclonal anti-centrosomin antibody (Vaizel-Ohayon and Schejter, 1999) at 1:400 dilution; a rabbit polyclonal anti-PAV-KLP serum Rb3301 at 1:100. Goat antimouse, anti-rat or anti-rabbit secondary antibodies coupled to fluorescein or rhodamine (Cappel, West Chester, PA, USA) were used at 1:600 dilution.…”