Kinesin-like Motor Protein Families› Kinesin-5› Kinesin-14

SpPkl1 Schizosaccharomyces pombe (fission yeast)

HsHSET Homo sapiens (Humans)

Leilani Cruz Final Report SURP

Bipolar Spindle Assembly

Mammalian spindle

Oligonucleotide Plasmid

› Insert› Vector

MTOC (Microtubule Organizing Center)

Paluh, J.L. (2008) Kinesin-14 leaps to pole position in bipolar spindle assembly. Chinese Journal of Cancer, 27(9): 1-5.

  Rodriguez, A.S., Killilea, A.N., Batac, J., Filopei, J.,

Simeonov, D.R., Lin, I., and J.L. Paluh (2008) Protein complexes at the microtubule organizing center regulate bipolar spindle assembly. Cell Cycle. 7(9): 1246-1253.

Simeonov, D.R., Kenny, K., Seo, L., Moyer, A., and J. L. Paluh. (2009) Distinct Kinesin-14 Mitotic Mechanisms in Spindle Bipolarity. Cell Cycle, 8 (21): 3571-583.

  Cruz, L. (2010) Defining Minimal Elements in a

Novel Kinesin-14 Tail Domain for Spindle Pole Localization and γ-TuSC Regulation. Summer Undergrad Research Fellowship (SURP) final report.

Kinesin-14 Pkl1 Tail domain, DD/35DA4B, contains elements for localization to spindle poles and functional interactions with MTOC proteins that are needed for it to regulate spindle bipolarity.

DD/

PCR (Polymerase Chain Reaction)› Oligonucleotides contain changes› PCR allows amplified copies of the altered

Tail region DNA, DD/35DA4B to be generated for cloning

Puc18 DD DD

Ligation Reaction› To join the two linear pieces of DNA: the

insert (DD/35DA4B) and the intermediate vector, Puc-18.

› The intermediate vector was used first to provide us with the correct cloning sites for later ligations.

Plasmid

Insert: DD/35DA4B

Vector: Puc-18

Bacterial Transformation› To separate products of the ligation

reaction:ie. successful clones from non-successful clones Bacterial

Cell

Vector

Insert

Plasmid

Quick Screen DNA Analysis› To identify the correct ligation product

amongst transformed bacterial colonies.

Leilani Cruz Final Report SURP

Correct clones, (DD/35DA4B Puc-18) were then cloned into the yeast vector: Prep81.

Identical experiments were repeated with the new vector Prep81continued through the Quick Screen DNA isolation procedure.

Yeast Transformation› To test the in vivo function of altered

Kinesin-14 proteins.

Leilani Cruz Final Report SURP

Serial Dilution Assay› Compare strains by plating decreasing cell

numbers of each onto triplicate plates and monitoring growth at different temperatures against wild type (positive) and nonfunctional (negative) gene controls.

In construct DD/35DA4B deletion of region D is minimal we predict that the construct will be functional.

DD/

Kinesin-14 Pkl1 Tail domain contains critical elements in order to regulate spindle assembly.

Construct DDP6 proved that part of region 6 and possibly D are critical and can not be deleted.

Construct 35DA4B proved that regions A, 4, and B are not critical and can be deleted.

Finish testing remaining constructs for functionality:› 3P56P› 3P6D› 3P56DC› DD 

Examine localization and protein interactions between modified Kinesin-14 Pkl1 and γ-TuSC MTOC proteins.

Paluh, J.L. (2008) Kinesin-14 leaps to pole position in bipolar spindle assembly. Chinese Journal of Cancer, 27(9): 1-5.

  Rodriguez, A.S., Killilea, A.N., Batac, J., Filopei, J., Simeonov, D.R., Lin, I.,

and J.L. Paluh (2008) Protein complexes at the microtubule organizing center regulate bipolar spindle assembly. Cell Cycle. 7(9): 1246-1253.

  Simeonov, D.R., Kenny, K., Seo, L., Moyer, A., and J. L. Paluh. (2009)

Distinct Kinesin-14 Mitotic Mechanisms in Spindle Bipolarity. Cell Cycle, 8 (21): 3571-583.

  Cruz, L. (2010) Defining Minimal Elements in a Novel Kinesin-14 Tail

Domain for Spindle Pole Localization and γ-TuSC Regulation. Summer Undergrad Research Fellowship (SURP) final report.

  Kollman, J.M., Zelter, A., Muller, E.G.D., Fox, B., Rice, L.M. Davis, T.N.,

and D.A. Agard (2008) The structue of the g-tubulin small complex: Implications of its architecture and flexibility for microtubule nucleation. Molec. Biol. Cell 19: 207-215.

Dr. Janet L. Paluh, CNSE at Univ. at Albany SUNY

  Leilani Cruz and Lonnie Seo, Rensselaer

Polytechnic Institute  Ms. Gleason and Ms. Strauss  My Family