Summary The microtubule motor Kinesin-1 plays central roles in intracellular transport. It has been widely assumed that many cellular cargos are moved by multiple Kinesins and that cargos with more motors move faster and for longer distances; concrete evidence, however, is sparse. Here we rigorously test these notions using lipid droplets in Drosophila embryos: We first employ antibody inhibition, genetics, biochemistry, and particle tracking to demonstrate that Kinesin-1 mediates plus-end droplet motion. We then measure how variation in Kinesin-1 expression affects the forces driving individual droplets and estimate the number of Kinesins actively engaged per droplet. Unlike in vitro, increased motor number results neither in longer travel distances nor higher velocities. Our data suggest that cargos in vivo can simultaneously engage multiple Kinesins and that transport properties are largely unaffected by variation in motor number. Apparently, higher-order regulatory mechanisms rather than motor number per se dominate cargo transport in vivo.

Molecular motor proteins are crucial for the proper distribution of organelles and vesicles in cells. Much of our current understanding of how motors function stems from studies of single motors moving cargos in vitro. More recently, however, there has been mounting evidence that the cooperation of multiple motors in moving cargos and the regulation of motor-filament affinity could be key mechanisms that cells utilize to regulate cargo transport. Here, we review these recent advances and present a picture of how the different mechanisms of regulating the number of motors moving a cargo could facilitate cellular functions.

Molecular motor proteins use the energy released from ATP hydrolysis to generate force and haul cargoes along cytoskeletal filaments. Thus, measuring the force motors generate amounts to directly probing their function. We report on optical trapping methodology capable of making precise in vivo stall-force measurements of individual cargoes hauled by molecular motors in their native environment. Despite routine measurement of motor forces in vitro, performing and calibrating such measurements in vivo has been challenging. We describe the methodology recently developed to overcome these difficulties, and used to measure stall forces of both kinesin-1 and cytoplasmic dynein-driven lipid droplets in Drosophila embryos. Critically, by measuring the cargo dynamics in the optical trap, we find that there is memory: it is more likely for a cargo to resume motion in the same direction-rather than reverse direction-after the motors transporting it detach from the microtubule under the force of the optical trap. This suggests that only motors of one polarity are active on the cargo at any instant in time and is not consistent with the tug-of-war models of bidirectional transport where both polarity motors can bind the microtubules at all times. We further use the optical trap to measure in vivo the detachment rates from microtubules of kinesin-1 and dynein-driven lipid droplets. Unlike what is commonly assumed, we find that dynein's but not kinesin's detachment time in vivo increases with opposing load. This suggests that dynein's interaction with microtubules behaves like a catch bond.

As our knowledge of biological processes advances, we are increasingly aware that cells actively position sub-cellular organelles and other constituents to control a wide range of biological processes. Many studies quantify the position and motion of, for example, fluorescently labeled proteins, protein aggregates, mRNA particles or virus particles. Both differential interference contrast (DIC) and fluorescence microscopy can visualize vesicles, nuclei or other small organelles moving inside cells. While such studies are increasingly important, there has been no complete analysis of the different tracking methods in use, especially from the practical point of view. Here we investigate these methods and clarify how well different algorithms work and also which factors play a role in assessing how accurately the position of an object can be determined. Specifically, we consider how ultimate performance is affected by magnification, by camera type (analog versus digital), by recording medium (VHS and SVHS tape versus direct tracking from camera), by image compression, by type of imaging used (fluorescence versus DIC images) and by a variety of sources of noise. We show that most methods are capable of nanometer scale accuracy under realistic conditions; tracking accuracy decreases with increasing noise. Surprisingly, accuracy is found to be insensitive to the numerical aperture, but, as expected, it scales with magnification, with higher magnification yielding improved accuracy (within limits of signal-to-noise). When noise is present at reasonable levels, the effect of image compression is in most cases small. Finally, we provide a free, robust implementation of a tracking algorithm that is easily downloaded and installed.

Neurons rely on microtubule (MT) motor proteins such as kinesin-1 and dynein to transport essential cargos between the cell body and axon terminus. Defective axonal transport causes abnormal axonal cargo accumulations and is connected to neurodegenerative diseases, including Alzheimer's disease (AD). Glycogen synthase kinase 3 (GSK-3) has been proposed to be a central player in AD and to regulate axonal transport by the MT motor protein kinesin-1. Using genetic, biochemical and biophysical approaches in Drosophila melanogaster, we find that endogenous GSK-3 is a required negative regulator of both kinesin-1-mediated and dynein-mediated axonal transport of the amyloid precursor protein (APP), a key contributor to AD pathology. GSK-3 also regulates transport of an unrelated cargo, embryonic lipid droplets. By measuring the forces motors generate in vivo, we find that GSK-3 regulates transport by altering the activity of kinesin-1 motors but not their binding to the cargo. These findings reveal a new relationship between GSK-3 and APP, and demonstrate that endogenous GSK-3 is an essential in vivo regulator of bidirectional APP transport in axons and lipid droplets in embryos. Furthermore, they point to a new regulatory mechanism in which GSK-3 controls the number of active motors that are moving a cargo.

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A new method of direct and continuous measurement of the spring constant of single molecule or molecular complex is elaborated. To that end the standard force spectroscopy technique with functionalized tips and samples is combined with a small dithering of the tip. The change of the dithering amplitude as a function of the pulling force is measured to extract the spring constant of the complex. The potentialities of this method are illustrated for the experiments with single bovine serum albumin-its polyclonal antibody (Ab-BSA) and fibrinogen-fibrinogen complexes.

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