Isolation and Purification of Kinesin from Drosophila Embryos

Summary

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Abstract

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 °C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Protocol

Strains of flies can be purchased and amplified in the lab. Depending on the amount of Drosophila culture vials received, one need to frequently 'flip' the culture vials, once the vials has reached maximum capacity. The amplification continues until approximately 50 Drosophila culture vials are filled. One then makes 'fly cups' with 100 ml tricorn beakers (Fly cup making is discussed in the next section). After 24 hours, use embryos from these cups to seed new Drosophila culture vials with affixed fly food at the bottom. At room temperature, the growth time for Drosophila embryos from overnight embryos to full grown flies are about ....

Discussion

Bovine brain is the most widely used starting material ¹¹ to purify full-length kinesin, though murine brain has been used as well ¹². A large disadvantage of using bovine brain as a kinesin source is the availability of fresh starting material: slaughterhouses are typically inaccessible, and the brain must be extremely fresh in order to obtain active kinesin. Further, only the brains of young cows are effective. Finally, genetic manipulation of cows is currently not a viable option, so only 'wild-t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar (Granulated)	Fisher Scientific	1423-500
Dextrose (D-Glucose) Anhydrous	Fisher Scientific	D16-3
Petri Dishes	BD Biosciences	35 1007	60x15mm Style (Polyester) 20/bag
Drosophila Culture Vials	UCI
Tricorn beaker	Econo Lab Inc.	B700-100	Volume:100ml, size: 58x72mm
Yeast	Red Star	2751
Nylon Mesh	Genesee Scientific	57-102	120 micron pore size
Nylon Mesh	Small Parts, Inc.	06-350/35
Pipes	Sigma-Aldrich	108321-27-3
Pipes	Sigma-Aldrich	108321-27-3
Glycerol	Fisher Scientific	56-81-5
EGTA	Sigma-Aldrich	67-42-5
MgS04(Anhydrous)	Fisher Scientific	7487-88-9
PMSF	Sigma-Aldrich	329-98-6
Leupeptin	Sigma-Aldrich	103476-89-7
Aprotonin	Sigma-Aldrich	9087-70-1
TAME	Sigma-Aldrich	178403-8
STI	Calbiochem	65635
GTP	Sigma-Aldrich	36051-31-7
Taxol	Sigma-Aldrich	33069-62-4
ATP analog 5’-adenylyl imidodiphosphate	Sigma-Aldrich	3605-31-7
NaCl	Mallinckrodt Baker Inc.	7647-14-5
ATP	Sigma-Aldrich	74804-12-9
Amicon Ultra Centrifugal Filters: .5 mL, 100kD cut off, 8 pack	EMD Millipore	UFC510008