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The interaction between cytoplasmic dynein and dynactin in intracellular transport / Sher Bahadur Karki.
LIBRA Thesis K18 1997
Available from offsite location
LIBRA Diss. POPM1997.317
Available from offsite location
- Format:
- Book
- Manuscript
- Microformat
- Thesis/Dissertation
- Author/Creator:
- Karki, Sher Bahadur.
- Language:
- English
- Subjects (All):
- Penn dissertations--Cell biology.
- Cell biology--Penn dissertations.
- Penn dissertations--Molecular biology.
- Molecular biology--Penn dissertations.
- Cell and Molecular Biology.
- Academic Dissertations as Topic.
- Medical Subjects:
- Cell and Molecular Biology.
- Academic Dissertations as Topic.
- Local Subjects:
- Penn dissertations--Cell biology.
- Cell biology--Penn dissertations.
- Penn dissertations--Molecular biology.
- Molecular biology--Penn dissertations.
- Physical Description:
- xi, 160 pages : illustrations ; 29 cm
- Production:
- 1997.
- Summary:
- Coordinated movement of organelles along the polarized array of microtubules is critical in diverse cellular processes such as mitosis, exocytosis, and axonal transport. Such directed motility is carried out by the microtubule motors, cytoplasmic dynein and kinesin. Cytoplasmic dynein is a minus-end-directed microtubule motor that requires another complex, dynactin, for vesicle motility in vitro. A number of genetic studies in Drosophila, yeast, and filamentous fungi suggest that dynein and dynactin overlap in function. However, the nature of dynein-dynactin interaction and the mechanism of dynactin function in dynein-mediated vesicular transport were not known. In this study we examined the interaction between cytoplasmic dynein and dynactin by affinity chromatography. Affinity columns of p150$\rm\sp{Glued}$ (a dynactin subunit) and dynein intermediate chains were constructed and the ability of these columns to retain dynein or dynactin from a brain homogenate was investigated. We found that the column-bound recombinant p150$\rm\sp{Glued}$ binds to the dynein complex and that dynein intermediate chains bind to the dynactin complex. We then hypothesized that this interaction may be essential in dynein-mediated vesicle transport. We tested our model in the well characterized extruded axoplasm from squid giant axon. We demonstrate that the anti-p150$\rm\sp{Glued}$ antibodies that disrupt dynein-dynactin interaction also inhibit the bi-directional organelle motility on microtubules and induce a complete dissociation of dynein from organelles. These observations support the hypothesis that dynactin may be required to form a translocation-competent ternary complex that consists of dynein, dynactin, and the organelle. We have also examined two small polypeptides that are retained by the dynein intermediate chain column. The 27 kDa polypeptide from a dynein affinity column was found to be identical in sequence to the $\beta$-subunit of the regulatory kinase Casein Kinase II (CKII). Although CKII is not an integral part of dynactin, evidence for the ability of CKII to phosphorylate dynein in vitro is presented. Finally, we have cloned the smallest dynactin subunit, p22, which was found to encode a novel, $\alpha$-helical polypeptide. Models for the function of dynactin in intracellular transport are presented.
- Notes:
- Supervisor: Erika L. F. Holzbaur.
- Thesis (Ph.D. in Cell and Molecular Biology) -- University of Pennsylvania, 1997.
- Includes bibliographical references.
- Local Notes:
- University Microfilms order no.: 98-14867.
- OCLC:
- 187457245
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