Journal of Cell Science, Vol 107, Issue 10 2839-2849, Copyright © 1994 by Company of Biologists

JOURNAL ARTICLES

Purification and characterization of ensconsin, a novel microtubule stabilizing protein

JC Bulinski and A Bossler
Department of Anatomy and Cell Biology, Columbia University, College of Physicians & Surgeons, New York, NY 10032.

In previous studies (Bulinski and Borisy (1979). Proc. Nat. Acad. Sci. 76, 293-297; Weatherbee et al. (1980). Biochemistry 19, 4116-4123) a microtubule-associated protein (MAP) of M(r) approximately 125,000 was identified as a prominent MAP in HeLa cells. We set out to perform a biochemical characterization of this protein, and to determine its in vitro functions and in vivo distribution. We determined that, like the assembly-promoting MAPs, tau, MAP2 and MAP4, the 125 kDa MAP was both proteolytically sensitive and thermostable. An additional property of this MAP; namely, its unusually tight association with a calcium-insensitive population of MTs in the presence of taxol, was exploited in devising an efficient purification strategy. Because of the MAP's tenacious association with a stable population of MTs, and because it appeared to contribute to the stability of this population of MTs in vitro, we have named this protein ensconsin. We examined the binding of purified ensconsin to MTs; ensconsin exhibited binding that saturated its MT binding sites at an approximate molar ratio of 1:6 (ensconsin:tubulin). Unlike other MAPs characterized to date, ensconsin's binding to MTs was insensitive to moderate salt concentrations (< or = 0.6 M). We further characterized ensconsin in immunoblotting experiments using mouse polyclonal anti-ensconsin antibodies and antibodies reactive with previously described MAPs, such as high molecular mass tau isoforms, dynamin, STOP, CLIP-170 and kinesin. These experiments demonstrated that ensconsin is distinct from other proteins of similar M(r) that may be present in association with MTs. Immunofluorescence with anti-ensconsin antibodies demonstrated that ensconsin was detectable in association with most or all of the MTs of several lines of human epithelial, fibroblastic and muscle cells; its in vivo properties and distribution, especially in response to drug or other treatments of cells, were found to be different from those of MAP4, the predominant MAP found in these cell types. We conclude that ensconsin, a MAP found in a variety of human cells, is biochemically - and perhaps functionally - distinct from other MAPs present in non-neuronal cells.

This article has been cited by other articles:

				G. Salpingidou, A. Smertenko, I. Hausmanowa-Petrucewicz, P. J. Hussey, and C. J. Hutchison A novel role for the nuclear membrane protein emerin in association of the centrosome to the outer nuclear membrane J. Cell Biol., September 7, 2007; 178(6): 897 - 904. [Abstract] [Full Text] [PDF]

				J. C. Bulinski, D. J. Odde, B. J. Howell, T. D. Salmon, and C. M. Waterman-Storer Rapid dynamics of the microtubule binding of ensconsin in vivo J. Cell Sci., January 11, 2001; 114(21): 3885 - 3897. [Abstract] [Full Text] [PDF]

				C. M. Waterman-Storer, W. C. Salmon, and E.D. Salmon Feedback Interactions between Cell-Cell Adherens Junctions and Cytoskeletal Dynamics in Newt Lung Epithelial Cells Mol. Biol. Cell, July 1, 2000; 11(7): 2471 - 2483. [Abstract] [Full Text]

				C. M. WATERMAN-STORER and E. D. SALMON Fluorescent speckle microscopy of microtubules: how low can you go? FASEB J, December 1, 1999; 13(9002): 225S - 230S. [Abstract] [Full Text] [PDF]

				K Faire, C. Waterman-Storer, D Gruber, D Masson, E. Salmon, and J. Bulinski E-MAP-115 (ensconsin) associates dynamically with microtubules in vivo and is not a physiological modulator of microtubule dynamics J. Cell Sci., January 12, 1999; 112(23): 4243 - 4255. [Abstract] [PDF]

				H. Nguyen, D Gruber, and J. Bulinski Microtubule-associated protein 4 (MAP4) regulates assembly, protomer-polymer partitioning and synthesis of tubulin in cultured cells J. Cell Sci., January 6, 1999; 112(12): 1813 - 1824. [Abstract] [PDF]

				S. S.L. Andersen and E. Karsenti XMAP310: A Xenopus Rescue-promoting Factor Localized to the Mitotic Spindle J. Cell Biol., November 17, 1997; 139(4): 975 - 983. [Abstract] [Full Text] [PDF]

				H. Nguyen, S Chari, D Gruber, C. Lue, S. Chapin, and J. Bulinski Overexpression of full- or partial-length MAP4 stabilizes microtubules and alters cell growth J. Cell Sci., January 1, 1997; 110(2): 281 - 294. [Abstract] [PDF]

				B. Eichenmuller, P. Everley, J. Palange, D. Lepley, and K. A. Suprenant The Human EMAP-like Protein-70 (ELP70) Is a Microtubule Destabilizer That Localizes to the Mitotic Apparatus J. Biol. Chem., January 4, 2002; 277(2): 1301 - 1309. [Abstract] [Full Text] [PDF]