“…The averages of velocities (Figure B) and run lengths (Figure C) of the small and large mitochondria in the cell body were independent of size. Importantly, the average velocity of ∼800 nm/second is typical of the unloaded velocity for both kinesin and dynein . Thus, in the cell body, the effect of drag appears negligible consistent with a previous lipid droplet study in drosophila embryos .…”
There is increasing interest in factors that can impede cargo transport by molecular motors inside the cell. While potentially relevant (1), the importance of cargo size and sub-cellular location have received relatively little attention. Here we address these questions taking advantage of the fact that mitochondria—a common cargo—in Drosophila neurons exhibit a wide distribution of sizes. In addition, the mitochondria can be genetically marked with GFP making it possible to visualize and compare their movement in the cell bodies and processes of living cells. Using total internal reflection (TIRF) microscopy coupled with particle tracking and analysis, we quantified transport properties of GFP positive mitochondria as a function of their size and location. In neuronal cell bodies we find little evidence for significant opposition to motion, consistent with a previous study on lipid droplets (2). However, in the processes we observe an inverse relationship between mitochondrial size and velocity and run distances. This can be ameliorated via hypotonic treatment to increase process size, suggesting that motor mediated movement is impeded in this more confined environment. Interestingly, we also observe local mitochondrial accumulations in processes but not in cell bodies. Such accumulations do not completely block transport, but do increase the probability of mitochondria-mitochondria interactions. They are thus particularly interesting in relation to mitochondrial exchange of elements.
“…The averages of velocities (Figure B) and run lengths (Figure C) of the small and large mitochondria in the cell body were independent of size. Importantly, the average velocity of ∼800 nm/second is typical of the unloaded velocity for both kinesin and dynein . Thus, in the cell body, the effect of drag appears negligible consistent with a previous lipid droplet study in drosophila embryos .…”
There is increasing interest in factors that can impede cargo transport by molecular motors inside the cell. While potentially relevant (1), the importance of cargo size and sub-cellular location have received relatively little attention. Here we address these questions taking advantage of the fact that mitochondria—a common cargo—in Drosophila neurons exhibit a wide distribution of sizes. In addition, the mitochondria can be genetically marked with GFP making it possible to visualize and compare their movement in the cell bodies and processes of living cells. Using total internal reflection (TIRF) microscopy coupled with particle tracking and analysis, we quantified transport properties of GFP positive mitochondria as a function of their size and location. In neuronal cell bodies we find little evidence for significant opposition to motion, consistent with a previous study on lipid droplets (2). However, in the processes we observe an inverse relationship between mitochondrial size and velocity and run distances. This can be ameliorated via hypotonic treatment to increase process size, suggesting that motor mediated movement is impeded in this more confined environment. Interestingly, we also observe local mitochondrial accumulations in processes but not in cell bodies. Such accumulations do not completely block transport, but do increase the probability of mitochondria-mitochondria interactions. They are thus particularly interesting in relation to mitochondrial exchange of elements.
“…It is also possible that the force‐based interaction between motors (not included in the theoretical model) could be altered by the nucleotide state of tubulin in microtubules. Lastly, although extensive in vitro studies suggest that this is unlikely (Block et al, ; Gelles et al, ; Xu et al, ), it is formally possible that polystyrene beads may alter the interactions between the motor and the microtubule in unexpected ways. Since it is challenging to completely rule out these potential concerns in biophysics‐based assays, we next turned to a biochemistry‐based assay.…”
Molecular motors such as kinesin-1 work in small teams to actively shuttle cargos in cells, for example in polarized transport in axons. Here we examined the potential regulatory role of the nucleotide state of tubulin on the run length of cargos carried by multiple kinesin motors, using an optical trapping-based in vitro assay. Based on a previous report that kinesin binds preferentially to GTP-tubulin-rich microtubules, we anticipated that multiple-kinesin cargos would run substantially greater distances along GMPCPP microtubules than along GDP microtubules. Surprisingly, we did not uncover any significant differences in run length between microtubule types. A combination of single-molecule experiments, comparison with previous theory, and classic microtubule affinity pulldown assays revealed that native kinesin-1 does not bind preferentially to GTP-tubulin-rich microtubules. The apparent discrepancy between our observations and the previous report likely reflects differences in post-translational modifications between the native motors used here and the recombinant motors examined previously. Future investigations will help shed light on the interplay between the motor’s post-translational modification and the microtubule’s nucleotide-binding state for transport regulation in vivo.
“…6,7 Thus, there is significant interest in achieving a mechanistic understanding of their singlemolecule function, and in relating this to ensemble function involving multiple motors. [8][9][10][11][12][13] In our single-molecule studies of kinesin-1, we observed surprising heterogeneity of function: many motors moved relatively rapidly (~800 nm/s), but some moved significantly slower (eg,~200 nm/s). We initially ignored this slower population as "unhealthy," and focused on the faster population.…”
Section: Introductionmentioning
confidence: 84%
“…Purification of Drosophila full‐length kinesin is as explained in Reference . To ensure that the population of kinesins are chemically “identical”, we expressed the functional, truncated kinesin (K560) and K‐560gfp in E. coli and purified it as reported earlier . The procedure briefly is, E. coli Rosetta cells were transformed, and grown at 37°C from a single colony in 500 mL of terrific broth.…”
Section: Methodsmentioning
confidence: 99%
“…They serve a wide range of cellular roles, and have a central role in creating and maintaining cellular organization—indeed, impaired function is linked to diseases such as neurodegeneration . Thus, there is significant interest in achieving a mechanistic understanding of their single‐molecule function, and in relating this to ensemble function involving multiple motors . In our single‐molecule studies of kinesin‐1, we observed surprising heterogeneity of function: many motors moved relatively rapidly (~800 nm/s), but some moved significantly slower (eg, ~200 nm/s).…”
The kinesin family proteins are often studied as prototypical molecular motors; a deeper understanding of them can illuminate regulation of intracellular transport. It is typically assumed that they function identically. Here we find that this assumption of homogeneous function appears incorrect: variation among motors' velocities in vivo and in vitro is larger than the stochastic variation expected for an ensemble of "identical" motors. When moving on microtubules, slow and fast motors are persistently slow, and fast, respectively. We develop theory that provides quantitative criteria to determine whether the observed single-molecule variation is too large to be generated from an ensemble of identical molecules. To analyze such heterogeneity, we group traces into homogeneous sub-ensembles. Motility studies varying the temperature, pH and glycerol concentration suggest at least 2 distinct functional states that are independently affected by external conditions. We end by investigating the functional ramifications of such heterogeneity through Monte-Carlo multi-motor simulations.
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