Search

Summary: Cilia are microtubule-based sensing hubs that rely on intraflagellar transport (IFT) for their development, maintenance, and function. Kinesin-2 motors transport IFT trains, consisting of IFT proteins and cargo, from ciliary base to tip. There, trains turn around and are transported back by IFT dynein. The mechanism of tip turnaround has remained elusive. Here, we employ single-molecule fluorescence microscopy of IFT components in the tips of phasmid cilia of living C. elegans. Analysis of the trajectories reveals that while motor proteins and IFT-A particle component CHE-11 mostly turn around immediately, the IFT-B particle component OSM-6 pauses for several seconds. Our data indicate that IFT trains disassemble into at least IFT-A, IFT-B, IFT-dynein, and OSM-3 complexes at the tip, where OSM-6 is temporarily retained or undergoes modification, prior to train reassembly and retrograde transport. The single-molecule approach used here is a valuable tool to study how directional switches occur in microtubule-based transport processes. : Using single-molecule fluorescence microscopy, Mijalkovic et al. visualize the dynamics of IFT components at the tips of C. elegans chemosensory cilia. They find that the motors and the IFT-A particle component CHE-11 reverse almost immediately, while the IFT-B component OSM-6 is temporarily retained before reassembly and reversal. Keywords: intracellular transport, IFT, kinesin, dynein, single-molecule biophysics, ciliary tip turns

Intraflagellar transport (IFT) in C. elegans chemosensory cilia is an example of functional coordination and cooperation of two motor proteins with distinct motility properties operating together in large groups to transport cargoes: a fast and processive homodimeric kinesin-2, OSM-3, and a slow and less processive heterotrimeric kinesin-2, kinesin-II. To study the mechanism of the collective dynamics of kinesin-II of C. elegans cilia in an in vitro system, we used Total Internal Reflection Fluorescence microscopy to image the motility of truncated, heterodimeric kinesin-II constructs at high motor densities. Using an analysis technique based on correlation of the fluorescence intensities, we extracted quantitative motor parameters, such as motor density, velocity and average run length, from the image. Our experiments and analyses show that kinesin-II motility parameters are far less affected by (self) crowding than OSM-3. Our observations are supported by numerical calculations based on the TASEP-LK model (Totally Asymmetric Simple Exclusion Process-Langmuir Kinetics). From a comparison of data and modelling of OSM-3 and kinesin-II, a general picture emerges of the collective dynamics of the kinesin motors driving IFT in C. elegans chemosensory cilia and the way the motors deal with crowding.

Molecular motor proteins fulfill the critical function of transporting organelles and other building blocks along the biopolymer network of the cell’s cytoskeleton, but crowding effects are believed to crucially affect this motor-driven transport due to motor interactions. Physical transport models, like the paradigmatic, totally asymmetric simple exclusion process (TASEP), have been used to predict these crowding effects based on simple exclusion interactions, but verifying them in experiments remains challenging. Here, we introduce a correlation imaging technique to precisely measure the motor density, velocity, and run length along filaments under crowding conditions, enabling us to elucidate the physical nature of crowding and test TASEP model predictions. Using the kinesin motor proteins kinesin-1 and OSM-3, we identify crowding effects in qualitative agreement with TASEP predictions, and we achieve excellent quantitative agreement by extending the model with motor-specific interaction ranges and crowding-dependent detachment probabilities. These results confirm the applicability of basic nonequilibrium models to the intracellular transport and highlight motor-specific strategies to deal with crowding.

Neuronal cells are highly crowded with various types of vesicles, organelles, proteins and cytoskeletal filaments densely packed inside the cytoplasm. One would expect crowding to pose challenges to motor-protein mediated transport, but in spite of the very crowded interior of the cell, motors appear to be able to deliver their cargo efficiently, overcoming traffic jams and roadblocks. The motors could be a possible source for crowding by creating traffic jams, limiting the number of motor-binding sites thus blocking the forward motion of motors. We use in vitro motility assays to take Total Internal Reflection Microscopy sequences of images of motors moving along microtubules. Here, we have developed an advanced analysis technique to obtain quantitative motor parameters from the image sequences in an automated way, using correlation of the fluorescence intensities. This new analysis method speeds up analysis and requires less data than the single-particle tracking approach. Correlating intensities allows analysis of data obtained under conditions inaccessible for single-particle tracking, including those performed at high motor densities that can provide insight into traffic-jam like interactions between motors. Apart from Kinesin-1 motors, this advanced analysis technique is used to understand the behavior of two kinesin-2 family motors in a crowded environment. These two kinesin-2 family motors, heterotrimeric kinesin-II and homodimeric OSM-3, cooperate to drive intraflagellar transport (IFT) in the sensory cilia of C. elegans. Our results based on the new analysis approach provide novel insights into the way kinesin motor proteins handle traffic-jam situations

Intracellular transport takes place in the crowded environment of the cell, densely packed with proteins, cytoskeletal filaments, vesicles and organelles. In most transport systems, multiple motors team up to transport a single cargo, resulting in collective motor behavior that cannot be explained only from single-motor properties. Intraflagellar transport (IFT) in C. elegans cilia is an example of functional coordination and cooperation of two motor proteins with distinct motility properties operating together in groups to transport a single cargo: homodimeric OSM-3 (fast and processive) and heterotrimeric Kinesin-II (slow and less processive). To study the mechanism of the collective dynamics of OSM-3 and kinesin-II in cilia, we use fluorescence microscopy and high-density in-vitro motility assays. Using an advanced analysis technique based on correlation of the fluorescence intensities, we extracted the quantitative motor parameters (such as motor density, velocity and average run length) from the image sequences in an automated way with a high level of precision. Our experiments and analyses show that OSM-3 and Kinesin-II motors are affected differently by the presence of the other motor. For example, OSM-3 velocity and run length are substantially reduced in the presence of the Kinesin-II. Numerical calculations based on TASEP-LK model (Totally Asymmetric Simple Exclusion Process-Langmuir Kinetics) underline the importance of intermolecular interactions, such as the change of the OSM-3 affinity to the microtubule dependent on the Kinesin-II density. Based on this approach, a general picture of collective dynamics of OSM-3 and Kinesin-II motor proteins in IFT is formulated.

An outstanding unresolved question is how does the mitotic spindle utilize microtubules and mitotic motors to coordinate accurate chromosome segregation during mitosis? This process depends upon the mitotic motor, kinesin-5, whose unique bipolar architecture, with pairs of motor domains lying at opposite ends of a central rod, allows it to crosslink microtubules within the mitotic spindle and to coordinate their relative sliding during spindle assembly, maintenance and elongation. The structural basis of kinesin-5’s bipolarity is, however, unknown, as protein asymmetry has so far precluded its crystallization. Here we use electron microscopy of single molecules of kinesin-5 and its subfragments, combined with hydrodynamic analysis plus mass spectrometry, circular dichroism and site-directed spin label electron paramagnetic resonance spectroscopy, to show how a staggered antiparallel coiled-coil ‘BASS’ (bipolar assembly) domain directs the assembly of four kinesin-5 polypeptides into bipolar minifilaments., During mitosis, kinesin-5 motors are thought to crosslink microtubules in a muscle-like sliding filament mechanism. By combining electron microscopy with other structural tools, the authors reveal how four kinesin-5 polypeptides are organized into bipolar minifilaments.

Heterotrimeric kinesin-2 motors [1] , [2] transport intraflagellar transport (IFT)-particles from the base to the tip of the axoneme to assemble and maintain cilia [3] , [4] , [5] , [6] , [7] , [8] , [9] , [10] . These motors are distinct in containing two non-identical motor subunits together with an accessory subunit [1] , [11] , [12] , [13] , [14] , [15] . We evaluated the significance of this organization by comparing purified wild type kinesin-2 holoenzymes that support IFT in vivo, with mutant trimers containing only one type of motor domain that do not support IFT in vivo. In motility assays, wild type kinesin-2 moved microtubules (MTs) at a rate intermediate between the rates supported by the two mutants. Interestingly, one of the mutants, but not the other mutant or the wild type protein, was observed to drive a persistent counter-clock-wise rotation of the gliding MTs. Thus one of the two motor domains of heterotrimeric kinesin-2 exerts torque as well as axial force as it moves along a MT, which may allow kinesin-2 to control its circumferential position around a MT doublet within the cilium.

Cilia are assembled and maintained by intraflagellar transport (IFT), the motor-dependent, bidirectional movement of multiprotein complexes, called IFT particles, along the axoneme. The sensory cilia of Caenorhabditis elegans represent very useful objects for studying IFT because of the availability of in vivo time-lapse fluorescence microscopy assays of IFT and multiple ciliary mutants. In this system there are 60 sensory neurons, each having sensory cilia on the endings of their dendrites, and most components of the IFT machinery operating in these structures have been identified using forward and reverse genetic approaches. By analyzing the rate of IFT along cilia within living wild-type and mutant animals, two anterograde and one retrograde IFT motors were identified, the functional coordination of the two anterograde kinesin-2 motors was established and the transport properties of all the known IFT particle components have been characterized. The anterograde kinesin motors have been heterologously expressed and purified, and their biochemical properties have been characterized using MT gliding and single molecule motility assays. In this chapter, we summarize how the tools of genetics, cell biology, electron microscopy, and biochemistry are being used to dissect the composition and mechanism of action of IFT motors and IFT particles in C. elegans.

Single-molecule fluorescence spectroscopy has become a crucial tool to study the behavior of biomolecules and their roles in complex biological processes. Single-molecule methods have in particular contributed to our understanding of the mechanism of force generation and motility of motor proteins. Motor proteins are enzymes that convert the chemical free energy obtained from the hydrolysis of adenosine triphosphate (ATP) into mechanical work in order to drive processes such as muscle contraction and intracellular transport. Here, we review how single-molecule fluorescence spectroscopy can be applied to motor proteins of the kinesin, myosin, and dynein superfamilies and how such studies have advanced our knowledge of the molecular basis of motor protein action.