Biochemical and immunocytochemical analysis of three novel microtubule-associated proteins in Drosophila melanogaster and the rat brain

Srinivasan, Shaila

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https://hdl.handle.net/2142/22689 Copy

Description

Title

Biochemical and immunocytochemical analysis of three novel microtubule-associated proteins in Drosophila melanogaster and the rat brain

Author(s)

Srinivasan, Shaila

Issue Date

1996

Doctoral Committee Chair(s)

Karr, Timothy L.

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Biology, Neuroscience
• Biology, Cell
• Chemistry, Biochemistry

Language

eng

Abstract

• Microtubules are essential organelles found in almost all eukaryotic cells. Some of their diverse functions include intracellular transport, cell division and maintenance of cell shape. Microtubules are composed of highly conserved proteins, the $\alpha$- and $\beta$-tubulins. Many of the functions associated with microtubules are thought to be due to the proteins that associate with them and are collectively termed microtubule-associated proteins or MAPs. Microtubule proteins (MTP), isolated from Drosophila embryos by repetitive cycles of assembly and disassembly, have been analyzed during embryonic development. The yield of twice-cycled MTP increased an average of 15-fold from 8 to 12 hours of development. Analysis of MTP purified from 6 and 16 hours after fertilization reveal differences in MAP composition with the 6 hour MTP having an increased number of putative MAPs compared to the 16 hour fraction.
• MTP from 12-16 hour embryo collections were used as antigen for the production of monoclonal antibodies (Mabs). Indirect immunofluorescence on whole embryos identified three hybridoma lines that stained cytoskeletal structures in the embryo, designated Mab DMAP45, DMAP55 and DMAP66. Mab DMAP45 recognizes a protein species with a My of 45 kD on immunoblots and in the embryo stains an actin-like pattern in the cytoplasm. DMAP45 also stains the developing ventral nerve cord. Mab DMAP55 predominantly recognizes a major band at 55 kD on immunoblots. Mab DMAP55 staining reveals structures present in spindle microtubules and the surrounding cytoplasm. Mab DMAP66 recognizes a single band of 66 kD on immunoblots and generally stains the cortical cytoplasm and in the yolk regions of the embryo. The staining patterns in the embryo and high-resolution electrophoretic analysis suggests that DMAP45 is $\gamma$-actin. In addition, Mab DMAP45 recognizes an additional protein species with the same molecular weight but a different pI suggesting that it may also recognize a posttranslational modification of actin not previously identified, one of recently discovered family of ARPs (actin-related proteins). DMAP55, based on its molecular weight and isoelectric point, may be a tubulin isoform. Although similar in molecular weight to tau protein, DMAP66 is not recognized by polyclonal antibodies against tau suggesting that it is a novel MAP not previously identified.
• Analyses of DMAP45, DMAP55 and DMAP66 proteins in rat brain tissue extracts and purified rat brain MTP identified proteins of almost identical molecular weights and isoelectric points. The presence of proteins with common biochemical properties in these widely divergent animal species suggests that they are related proteins that have been evolutionarily conserved.

Type of Resource

text

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Copyright 1996 Srinivasan, Shaila

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