Simple non-fluorescent polarity labeling of microtubules for molecular motor assays

Abstract

Transport of intracellular organelles along the microtubule cytoskeleton occurs in a bidirectional manner due to opposing activity of microtubule-associated motor proteins of the kinesin and dynein families. Regulation of this opposing activity and the resultant motion is believed to generate a polarized distribution of many organelles within the cell. The bidirectional motion can be reconstituted on in vitro assembled microtubules using organelles extracted from cells. This provides an opportunity to understand the regulation of intracellular transport through quantitative analysis of the motion of organelles in a controlled environment. Such analysis requires the use of polarity-labeled microtubules to resolve the plus and minus components of bidirectional motion. However, existing methods of in vitro microtubule polarity labeling are unsuitable for high-resolution recording of motion. Here we present a simple and reliable method that uses avidin-coated magnetic beads to prepare microtubules labeled at the minus end. The microtubule polarity can be identified without any need for fluorescence excitation. We demonstrate video-rate high-resolution imaging of single cellular organelles moving along plus and minus directions on labeled microtubules. Quantitative analysis of this motion indicates that these organelles are likely to be driven by multiple dynein motors in vivo.

Tug-of-war between dissimilar teams of microtubule motors regulates transport and fission of endosomes

“…When assayed with an optical trap, 54% of minus-moving endosomes could not escape from the trap and exhibited forces between 4 and 6 pN. When released from the trap, these endosomes moved smoothly over a long distance (Ͼ 20 m; Table S1; also see ref 18), typical of motion driven by four or more dyneins (22). These observations indicate that stall force of a single Dictyostelium dynein is significantly lower than Ϸ7 pN reported for yeast dynein coated onto beads (23).…”

“…Motion was observed in vitro on single MTs that were polarity labeled at the minus end with nonfluorescent magnetic beads (18). This allowed us to resolve the plus/minus components of motion (and thereby the activity of kinesin/dynein) in video tracks of moving endosomes with high temporal and spatial resolution.…”

“…Preparation of a crude extract containing motile endosomes was essentially the same as reported elsewhere (15,18). Motility was assayed at 22°C in a flow cell (8) with 0.5 L PNS added to a motility mixture [18.5 L of LB/15% sucrose and 1 L of a 20ϫ ATP regenerating mix (ϭ 20 mM ATP, 20 mM MgCl 2, 40 mM creatine phosphate, and 40 units/mL creatine kinase)].…”

“…Motility was assayed at 22°C in a flow cell (8) with 0.5 L PNS added to a motility mixture [18.5 L of LB/15% sucrose and 1 L of a 20ϫ ATP regenerating mix (ϭ 20 mM ATP, 20 mM MgCl 2, 40 mM creatine phosphate, and 40 units/mL creatine kinase)]. MTs were polarity labeled using magnetic avidin-coated beads (18). Briefly, short biotinylated MT seeds were preferentially extended from the plus end with a mixture of normal (nonbiotin) tubulin and N-ethylmaleimide (NEM) tubulin and adhered to a polylysine-coated coverslip.…”

“…Each pixel measured 98 ϫ 98 nm. Motion of single endosomes was tracked offline (37) with subpixel resolution (Ϸ5 nm) by calculating the centroid of a cross-correlation image and analyzed as described (18,22,25).…”