The term Kinesin refers to any motor protein that is often found in eukaryotic cells. “The term defines a broad class of motor proteins which move along microtubule cables powered by the hydrolysis of ATP.” (Rogers 2010) This active movement of “Kinesins supports several cellular functions which include mitosis, meiosis, and transport of other materials such as axonal transport.” (Takemura 2004) The proteins provide “homodimeric catastrophe factors in which the kinesin motor domain, which contains the MT- and ATP binding sites, lies in the middle of the amino acid sequence.” (Berriman et al., 2002 ) Kinesin 45 is thought to play a role in the chromosome segregation in Trypanosoma brucei, “an African trypanosome which infects a wide range of mammals including humans.” (Moores 2006) This study aims to localize Kinesin 45 within a trypanosome cell and identify its role during the mitotic process.
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Trypanosoma brucei is an extracellular parasite, it inhabits the intercellular space and in the bloodstream of its mammalian host. Its genome consists of three chromosome types which are characterized by their sizes. “There are 11 pairs of large (1 to >6Mbp) megabase sized chromosomes (MBCs, a variable number (1 to 5) of intermediate (200 to 900 Kb) sized chromosomes (ICs) and a population in the order of approximately 100 minichromosomes (50 to 150 Kb, MCs)” (Berriman et al., 2002) The trypanosomes show several differences from the metazoan kind of mitosis. They do not go through the observable chromosome condensation during the mitotic phase. Similar to yeast, they “perform a ‘closed mitosis’ where the mitotic spindle forms within the nucleus without the breakdown of its nuclear envelope.” (Takemura 2004)The T. brucei possesses about 120 chromosomes, for these chromosomes to segregate, the spindles in the mitotic phase should have at least “240 microtubules.” (Ogbadoyi et al., 2000) However, previous studies indicate that the number of microtubules present during mitosis does not exceed “100 which is well below the minimum requirement.” (Ogbadoyi et al., 2000)) Thus trypanosomes should be having an alternative method of chromosome segregation. Several theoretical models have been suggested to explain the possible methods of chromosome segregation in the T. brucei. A model of “minichromosome segregation called the ‘lateral stacking’ in which the MBCs and ICs are segregated conventionally while the associate laterally pole-to-pole microtubules of the mitotic spindle.” (Rogers 2010) This model gives a possible explanation for the chromosome segregation in the trypanosomes; it however gives little indication of the molecular mechanisms involved. This study partly aims at revealing the role “played by members of the kinesin-13 family, particularly Kinesin 45 in the chromosome segregation in T. brucei.” (Moores 2006) These kinesins have previously been associated with mitotic functions as they are microtube deployments. T. brucei posses 7 kinesins 13 family members, “Kinesin 45 (kif45) do not possess a positively charged neck domain and its isoelectric point has been calculated to 4.61,” thus it may have a different mitotic function compared to other members even though this characteristic can be seen in other members such as kif24. (Vanstraelen et al., 2006)
Materials and Methods
The kinesin 45 genes and the resultant protein sequences will be sourced from the “Trypanosoma brucei gene database. The sequences will be verified by the presence of kinesin motor domain as predicted by protein database.” (Letunic et al., 2006)
T. brucei culture
The procyclic strains of T. brucei (427, 449, and 29-13) will be maintained using the SDM-79 medium at 28 degrees Celsius. The bloodstream strain 442 will be grown in the HMI-9 medium at 37 degrees Celsius. “Hygromycin, phleomycin, and blasticidin antibiotics” will be used in the preservation and selection of the cell lines. (Wirtz et al., 1999) Transfection will be performed on the procyclic strains which will then be allowed to recuperate in SDM- 79 at 28 degrees Celsius.
Generation of 29-13/p2T7-TbKif45 RNAi cell lines
The vector p2T7-177 will be used to make the kinesin 45 constructs to allow “inducible production of double-stranded RNA (dsRNA) from opposing T7 promoters within the vector.” (Inoue et al., 1990) The targeted Tbkif45 genes will be amplified using a standard PCR. After suitable cloning, the cells will be transformed into competent “E. coli XLI-Blue cells for amplification.” (Takemura 2004) After overnight incubation at 37 degrees Celsius using the “Luria-Bertani media” the positive colonies will be selected and the DNA harvested via alkaline lysis. (Inoue et al., 1990) This will be followed by digestion using the restriction enzymes HindIII and BamHI and separation by agarose gel.
Making polyclonal antibody of Tbkif45
Protein expression vectors will be constructed using “the pTrcHisC vector which adds a polyhistidine tag at the N-terminus of the inserted gene fragment.” (wickstead et al., 2002) The DNA coding region for the kinesin 45 will be amplified using standard PCR using strain 427 as the template. The correct reading frame will be verified by sequencing the 100 bp upstream from the point of insertion of the PCR fragment. After the verification, the constructs will be used to express recombinant kinesin 45 fragments in the E.coli BL21 cell line. The transformed cells will be grown in a prewarmed LB medium at 37 degrees Celsius. “Isopropyl-beta-D-thiogalactopyranoside (IPTG)” will be added to the culture to induce the production of recombinant proteins. (wickstead et al., 2002) The cells will be harvested and “lysed via passaging through the French pressure cell; the resulting lysate will be centrifuged to separate the soluble from the insoluble fraction of the lysate.” (wickstead et al., 2002) For the soluble fraction, the recombinant proteins were collected by purification. While for the insoluble fraction the recombinant protein will be collected by discarding the supernatant, resuspending the pellet in a denaturing lysis buffer to solubilize. This will be followed by centrifugation to remove any particles.
SDS-polyacrylamide gel electrophoresis and western blotting
The entire procedure will be carried out at room temperature. The cells will be washed first once with PBS before being dissolved in “hot SDS PAGE loading buffer (0.045 M tris-Cl, pH 6.8, 10% glycerol, 1% SDS, 0.02% bromophenol blue, 2.5%beta-mercaptoethanol). The sample will then be loaded onto a 10% Tris/Glycine SDS-Polyacrylamide gel for protein separation.” (Sambrook 1989) The gel will then be transferred to a nitrocellulose membrane in “a NUpage buffer for one hour at 30 V. The membranes will be blocked using BSA or skimmed milk at 7% (w/v) and diluted in Tris-buffered saline containing 0.1% tween-20 (TBS-T) for about” 30 minutes at room temperature while shaking. (wickstead et al., 2002)The antibodies will be incubated “at 7% (w/v) milk TBS-, the membrane will be incubated first with the primary antibody for 1 hour, followed by washes with TBS-T before proceeding to incubation with a corresponding conjugated antibody for 1 hour.” (Moores 2006) The membrane will then be washed and processed using a chemiluminescence reagent and finally detected by wrapping the blots to a light-sensitive film for approximately 5 minutes.
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Cloning of TbKif45 and construction of Tbkif45myc cell line
This is the final step and will be done using tetracycline-inducible expression plasmids. The open reading frames for the kinesin 45 genes “will be amplified using the proofreading Accusure Taq polymerase on genomic DNA from a suitable trypanosome strain.” (Sambrook 1989)
Berriman, M., Hall, N., Sheader, K., Bringuad, F., and G. Rudenko. (2002) The architecture of variant surface glycoprotein gene expression sites in Trypanasoma brucei. Mol Biochem Parasitol. 122:131-40
Inoue, H., Nojima, H., and Okayama, H. (1990) High efficiency transformation of the Escherichia coli with plasmids. Gene. 96:23-8
Letunic, I., Copley, R., Pils, B., and Bork, P. (2006) SMART 5: domains in the context of genomes and networks. Nucleic Acids Res. 34: D257-60
Moores, C. (2006). Microtube depolymerisation by kinesin-13 motors. London: The Scripps Research institute.
Ogbadoyi, E., Ersfeld, K., Robinson, D., Sherwin, T., and Gull, K. (2000) Architecture of Trypanosoma Brucei nucleus during interphase and mitosis. Chromosoma. 108:501-13.
Rogers, D. S. (2010). Microtubule motors in mitosis. California: University of California.
Sambrook, J., fritsch, E and Maniatis, T. (1989) Molecular Cloning: A laboratory Manual
Takemura, N. H. (2004). Kinesin superfamily proteins and their various functions and dynamics. Web.
Vanstraelen, M., Inze, D., and Geelen, D. (2006) Mitosis-specific Kinesins in Arabidopsis. Trends Plants Sci. 11: 167-75.
Wickstead, B., Ersfeld, K., and Gull, K. (2002) Targeting of tetracycline-inducible expression system to the transcriptionally silent minichromosomes of Trypanosoma brucei. Mol biochem Parasitol. 125: 211-6
Wirtz, E., Leal, C., Ochart, C., and Cross, G. (1999) A tightly regulated inducible expression system for conditional gene Knock-outs and dominant-negative genetics in Trypanosoma Brucei. Mol Biochem Parasitol. 99:89-101