Identification of ubiquitous high-molecular-mass, heat-stable microtubule-associated proteins (MAPs) that are related to the Drosophila 205-kDa MAP but are not related to the mammalian MAP-4.

Abstract

Identification of ubiquitous high-molecular-mass, heat-stable microtubule-associated proteins (MAPs) The role of microtubules in a variety of cellular processes appears to be regulated, in part, by the many proteins that associate with microtubules. Microtubule-associated proteins (MAPs) have been characterized in a number of different systems and represent a diverse group ofproteins that are generally classified according to their apparent molecular masses or biochemical properties (for reviews, see refs. 1-4). The MAP-2, MAP-4, and T classes include proteins that are fibrous in structure and are stable at elevated temperatures. Of these, the largest number of proteins so far described has been in the MAP-4 class, members of which range from 180 to 240 kDa and which includes mouse MAP-4 (5), HeLa 210-kDa MAP (6, 7), Drosophila 205-kDa MAP (8), the 190-kDa MAPs identified in bovine adrenal gland (9) and rat liver (10), and rat MAP-3 (11). Complete or partial DNA sequence has now been reported for the genes encoding four of the MAP-4 class of proteins (12-15). Comparison of the predicted amino acid sequences of the HeLa, bovine, and mouse MAP-4 proteins has shown an overall sequence identity of -70%; the conserved residues of these three proteins are clustered in four regions, one of which includes the microtubule-binding domain (15). In contrast, the predicted amino acid sequence of the Drosophila 205-kDa MAP is not similar to either the mammalian MAP-4 proteins or to any other MAP for which sequence data is available (12).Using a monoclonal antibody (mAb; AX3) raised against microtubule-organizing centers from the cellular slime mold Dictyostelium discoideum, we have identified a highmolecular mass, heat-stable MAP that occurs in species ranging from Dictyostelium to humans (16). We show here that the proteins recognized by mAb AX3 in mouse and human are distinct from the previously identified MAP-2 and MAP-4 proteins. However, the AX3 antigens in Dictyostelium and mammals are immunologically related to the 205-kDa MAP of Drosophila, suggesting that the 205-kDa MAP and the AX3 antigens define a distinct class of heat-stable MAPs. MATERIALS AND METHODSAntibodies. The AX3 mAb was raised against an isolated nucleus/nucleus-associated body (the primary microtubuleorganizing center of Dictyostelium) fraction from the Ax-3 strain of Dictyostelium discoideum. The production and preliminary characterization of this mAb are described elsewhere (16).The antibodies against mouse MAP-4, HeLa 210-kDa MAP, Drosophila 205-kDa MAP, and mouse MAP-2 used in this study were provided by J. B. Olmsted (17), G. G. Borisy (6), L. S. B. Goldstein (8), and L. I. Binder (University of Alabama, Birmingham), respectively. Anti-tubulin antibodies used were either an antibody raised against yeast tyrosinated a-tubulin (from J. V. Kilmartin) (18) or an anti-chicken (-tubulin mAb (Amersham).Cell Culture and Protein Fractionation. Dictyostelium cells grown in suspension were collected by centrifugation and resuspended in 8-10 vol of homogeni...

CLAMP, a novel microtubule-associated protein with EB-type calponin homology

“…Immunoelectron microscopy data demonstrated a particularly dense association of 205 kDa MAP on the periphery of MT bundles rather than within the compact arrays, raising the issue as to whether this protein is a significant component of the cross bridges between actin microfilaments and MTs in these structures [Oshima et al, 1992]. The 205 kDa MAP is the homologue of the Drosophila 205 kDa MAP, which is a distinct class of heat-stable MAPs [Goldstein et al, 1986] that were shown to be expressed in murine cell lines [Kimble et al, 1992]. In contrast, we observed immunolabeling of CLAMP throughout the pillar cells and within the compact MT bundles, demonstrating that it is associated with MTs and may possibly function to crosslink and bundle these MTs [Tolomeo and Holley, 1997].…”

The Opitz syndrome gene product, MID1, associates with microtubules

“…HeLa microtubules were prepared from 4 ϫ 10 7 cells according to a protocol described previously (19) by using Taxol to facilitate microtubule assembly. MID1͞GFP-overexpressing COS-7 cells (1 ϫ 10 6 ) were treated with 200 ng͞ml colcemid overnight to destroy the microtubules, homogenized in 5 vol of microtubule assembly buffer (19), and centrifuged at 60,000 ϫ g for 60 min. The supernatant was mixed with Taxol, GTP, and HeLa microtubules and incubated at 37°C for 30 min.…”

The Opitz syndrome gene product MID1 assembles a microtubule-associated ribonucleoprotein complex

“…Microtubules were polymerised in vitro from 3 × 10 7 HeLa cells according to previously reported protocols (Kimble et al 1992; Vallee 1982) at 37°C. As control, a sample was continuously maintained under cold-shock conditions.…”