Intraflagellar transport (IFT), the motor-dependent movement of IFT particles along the axoneme, is critical for the assembly, maintenance, and function of motile and sensory cilia, and, consequently, this process underlies ciliary motility, cilium-based signaling, and ciliopathies. Here, I present my perspective on IFT as a model system for studying motor-driven cargo transport. I review evidence that kinesin-2 motors physically transport IFT particles as cargo and hypothesize that several accessory kinesins confer cilia-specific functions by augmenting the action of the two core IFT motors, kinesin-2 and dynein 1b, which assemble the cilium foundation.

Reaction coordinate diagrams are used to relate the free energy changes that occur during the progress of chemical processes to the rate and equilibrium constants of the process. Here I briefly review the application of these diagrams to the thermodynamics and kinetics of the generation of force and motion by cytoskeletal motors and polymer ratchets as they mediate intracellular transport, organelle dynamics, cell locomotion, and cell division. To provide a familiar biochemical context for discussing these subcellular force generators, I first review the application of reaction coordinate diagrams to the mechanisms of simple chemical and enzyme-catalyzed reactions. My description of reaction coordinate diagrams of motors and polymer ratchets is simplified relative to the rigorous biophysical treatment found in many of the references that I use and cite, but I hope that the essay provides a valuable qualitative representation of the physical chemical parameters that underlie the generation of force and motility at molecular scales. In any case, I have found that this approach represents a useful interdisciplinary framework for understanding, researching, and teaching the basic molecular mechanisms by which motors contribute to fundamental cell biological processes.

The mitotic spindle uses dynamic microtubules and mitotic motors to generate the pico-Newton scale forces that are needed to drive the mitotic movements that underlie chromosome capture, alignment and segregation. Here, we consider the biophysical and molecular basis of force-generation for chromosome movements in the spindle, and, with reference to the Drosophila embryo mitotic spindle, we briefly discuss how mathematical modeling can complement experimental analysis to illuminate the mechanisms of chromosome-to-pole motility during anaphase A and spindle elongation during anaphase B.

Chromosome segregation during anaphase depends on chromosome-to-pole motility and pole-to-pole separation. We propose that in Drosophila embryos, the latter process (anaphase B) depends on a persistent kinesin-5-generated interpolar (ip) microtubule (MT) sliding filament mechanism that "engages" to push apart the spindle poles when poleward flux is turned off. Here we investigated the contribution of the midzonal, antiparallel MT-cross-linking nonmotor MAP, Feo, to this "slide-and-flux-or-elongate" mechanism. Whereas Feo homologues in other systems enhance the midzone localization of the MT-MT cross-linking motors kinesin-4, -5 and -6, the midzone localization of these motors is respectively enhanced, reduced, and unaffected by Feo. Strikingly, kinesin-5 localizes all along ipMTs of the anaphase B spindle in the presence of Feo, including at the midzone, but the antibody-induced dissociation of Feo increases kinesin-5 association with the midzone, which becomes abnormally narrow, leading to impaired anaphase B and incomplete chromosome segregation. Thus, although Feo and kinesin-5 both preferentially cross-link MTs into antiparallel polarity patterns, kinesin-5 cannot substitute for loss of Feo function. We propose that Feo controls the organization, stability, and motor composition of antiparallel ipMTs at the midzone, thereby facilitating the kinesin-5-driven sliding filament mechanism underlying proper anaphase B spindle elongation and chromosome segregation.

Elongation of the mitotic spindle during anaphase B contributes to chromosome segregation in many cells. Here, we quantitatively test the ability of two models for spindle length control to describe the dynamics of anaphase B spindle elongation using experimental data from Drosophila embryos. In the slide-and-flux-or-elongate (SAFE) model, kinesin-5 motors persistently slide apart antiparallel interpolar microtubules (ipMTs). During pre-anaphase B, this outward sliding of ipMTs is balanced by depolymerization of their minus ends at the poles, producing poleward flux, while the spindle maintains a constant length. Following cyclin B degradation, ipMT depolymerization ceases so the sliding ipMTs can push the poles apart. The competing slide-and-cluster (SAC) model proposes that MTs nucleated at the equator are slid outward by the cooperative actions of the bipolar kinesin-5 and a minus-end-directed motor, which then pulls the sliding MTs inward and clusters them at the poles. In assessing both models, we assume that kinesin-5 preferentially cross-links and slides apart antiparallel MTs while the MT plus ends exhibit dynamic instability. However, in the SAC model, minus-end-directed motors bind the minus ends of MTs as cargo and transport them poleward along adjacent, parallel MT tracks, whereas in the SAFE model, all MT minus ends that reach the pole are depolymerized by kinesin-13. Remarkably, the results show that within a narrow range of MT dynamic instability parameters, both models can reproduce the steady-state length and dynamics of pre-anaphase B spindles and the rate of anaphase B spindle elongation. However, only the SAFE model reproduces the change in MT dynamics observed experimentally at anaphase B onset. Thus, although both models explain many features of anaphase B in this system, our quantitative evaluation of experimental data regarding several different aspects of spindle dynamics suggests that the SAFE model provides a better fit.

Intraflagellar transport (IFT) is a form of motor-dependent cargo transport that is essential for the assembly, maintenance, and length control of cilia, which play critical roles in motility, sensory reception, and signal transduction in virtually all eukaryotic cells. During IFT, anterograde kinesin-2 and retrograde IFT dynein motors drive the bidirectional transport of IFT trains that deliver cargo, for example, axoneme precursors such as tubulins as well as molecules of the signal transduction machinery, to their site of assembly within the cilium. Following its discovery in Chlamydomonas, IFT has emerged as a powerful model system for studying general principles of motor-dependent cargo transport and we now appreciate the diversity that exists in the mechanism of IFT within cilia of different cell types. The absence of heterotrimeric kinesin-2 function, for example, causes a complete loss of both IFT and cilia in Chlamydomonas, but following its loss in Caenorhabditis elegans, where its primary function is loading the IFT machinery into cilia, homodimeric kinesin-2-driven IFT persists and assembles a full-length cilium. Generally, heterotrimeric kinesin-2 and IFT dynein motors are thought to play widespread roles as core IFT motors, whereas homodimeric kinesin-2 motors are accessory motors that mediate different functions in a broad range of cilia, in some cases contributing to axoneme assembly or the delivery of signaling molecules but in many other cases their ciliary functions, if any, remain unknown. In this review, we focus on mechanisms of motor action, motor cooperation, and motor-dependent cargo delivery during IFT.

Abstract Background In the Drosophila melanogaster syncytial blastoderm stage embryo anaphase B is initiated by a cell cycle switch in which the suppression of microtubule minus end depolymerization and spatial reorganization of the plus ends of outwardly sliding interpolar microtubules triggers spindle elongation. RNA interference in Drosophila cultured S2 cells may present a useful tool for identifying novel components of this switch, but given the diversity of spindle design, it is important to first determine the extent of conservation of the mechanism of anaphase B in the two systems. Results The basic mechanism, involving an inverse correlation between poleward flux and spindle elongation is qualitatively similar in these systems, but quantitative differences exist. In S2 cells, poleward flux is only partially suppressed and the rate of anaphase B spindle elongation increases with the extent of suppression. Also, EB1-labelled microtubule plus ends redistribute away from the poles and towards the interpolar microtubule overlap zone, but this is less pronounced in S2 cells than in embryos. Finally, as in embryos, tubulin FRAP experiments revealed a reduction in the percentage recovery after photobleaching at regions proximal to the pole. Conclusions The basic features of the anaphase B switch, involving the suppression of poleward flux and reorganization of growing microtubule plus ends, is conserved in these systems. Thus S2 cells may be useful for rapidly identifying novel components of this switch. The quantitative differences likely reflect the adaptation of embryonic spindles for rapid, streamlined mitoses.

Anaphase B spindle elongation is characterized by the sliding apart of overlapping antiparallel interpolar (ip) microtubules (MTs) as the two opposite spindle poles separate, pulling along disjoined sister chromatids, thereby contributing to chromosome segregation and the propagation of all cellular life. The major biochemical "modules" that cooperate to mediate pole-pole separation include: (i) midzone pushing or (ii) braking by MT crosslinkers, such as kinesin-5 motors, which facilitate or restrict the outward sliding of antiparallel interpolar MTs (ipMTs); (iii) cortical pulling by disassembling astral MTs (aMTs) and/or dynein motors that pull aMTs outwards; (iv) ipMT plus end dynamics, notably net polymerization; and (v) ipMT minus end depolymerization manifest as poleward flux. The differential combination of these modules in different cell types produces diversity in the anaphase B mechanism. Combinations of antagonist modules can create a force balance that maintains the dynamic pre-anaphase B spindle at constant length. Tipping such a force balance at anaphase B onset can initiate and control the rate of spindle elongation. The activities of the basic motor filament components of the anaphase B machinery are controlled by a network of non-motor MT-associated proteins (MAPs), for example the key MT cross-linker, Ase1p/PRC1, and various cell-cycle kinases, phosphatases, and proteases. This review focuses on the molecular mechanisms of anaphase B spindle elongation in eukaryotic cells and briefly mentions bacterial DNA segregation systems that operate by spindle elongation.

The mechanical events of mitosis depend on the action of microtubules and mitotic motors, but whether these spindle components act alone or in concert with a spindle matrix is an important question.