Biomod/2012/Columbia/KinesinKings/Background

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Kinesin Kings Test


Contents

Definitions

Microtubule

Cross section view of a microtubule
Cross section view of a microtubule

Microtubules are an essential component of the cytoskeleton and are found in almost all eukaryotic cells. They are used in the development and maintenance of the shape of the cell, in vesicle transport in mitochondria, cell signalling, and perhaps most importantly, cell division and mitosis. Microtubules are made up of hollow cylinders made up of alpha,beta tubulin heterodimers. Tubulin dimers bind head to head to form linear protofilaments. Typically, a microtubule is composed of 13 (although it varies from 10 to 18) such protofilaments arranged as a cylinder, with a diameter of approximately 25 nm, and a length of many micrometers. Microtubules exhibit structural polarity as protofilaments bind with others in their same orientation, where the exposed alpha tubulin end is slow growing and called the minus end, and the exposed beta tubulin end is fast growing and named the plus end. While polymerizing, microtubules exhibit dynamic instability in that, when the microtubule is growing, the dimers in the plus end have a GTP molecule bound to them, which will be hydrolyzed to GDP. If the rate of polymerization is greater than that of hydrolysis, a ‘cap’ of GTP will form at the plus end, and stabilize the microtubule. Otherwise, the GTP cap is eventually destroyed and rapid depolymerization of the microtubule occurs, until a GTP tubulin is reached in the lattice of the microtubule, restarting the stable growth phase. To ensure a more static microtubule structure, one can suppress depolymerization by adding pacitexel (taxol) to the buffer solution, or an even stronger option is to chemically cross link microtubules using gluteraldehyde. In order to observe the microtubules in vitro, rhodamine dye can be used in conjunction with the tubulin prior to polymerization so that the assembled microtubules will be observed using fluorescence microscopy. Research involving microtubule assembly and structure has resulted in advancements in fields ranging from cancer therapy to selective cargo loading and transportation on the micrometer scale.

Polylyisine

Bonding of poly-L-lysine and glass surface
Bonding of poly-L-lysine and glass surface

Polylysine (poly-L-lysine) is a natural polymer of the essential amino acid lysine. It can be as long as a couple hundred nanometers, with a diameter of .1 - .2 nm, and has a positive charge. It is used to coat tissue culturerware as it increases cell adherency.

Current Practices

Scientists use in vitro motility assays to study the interaction between biomolecular motors (kinesin) and their corresponding filaments (microtubules). One category of motilty assay is the “gliding geometry” which keeps the motors stationary, and the filaments moving or ‘gliding’, inside simple flow cells made from a glass slide and coverslips seperated by adhesive tape, forming a flow chamber with a height of approximately 100 micrometers. Within the flow chamber, up until now, all microtubule arrangements have occurred in a direction parallel to both the horizontal surfaces and the flow direction. This geometry has proved useful in the construction of cargo transport pathways, however there is yet to be an arrangement of microtubules within the flow cell that would allow for the possibility of a micro-filtration device that would oppose incoming flow profiles in a perpendicular direction.


Kinesin Kings Test

Columbia

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