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Protein lateral mobility in cell membranes is generally measured using fluorescence photobleaching recovery (FPR). Since the development of this technique, the data have been interpreted by assuming free Brownian diffusion of cell surface receptors in two dimensions, an interpretation that requires that a subset of the diffusing species remains immobile. The origin of this so-called immobile fraction remains a mystery. In FPR, the motions of thousands of particles are inherently averaged, inevitably masking the details of individual motions. Recently, tracking of individual cell surface receptors has identified several distinct types of motion (Gross and Webb, 1988; Ghosh and Webb, 1988, 1990, 1994; Kusumi et al. 1993; Qian et al. 1991; Slattery, 1995), thereby calling into question the classical interpretation of FPR data as free Brownian motion of a limited mobile fraction. We have measured the motion of fluorescently labeled immunoglobulin E complexed to high affinity receptors (Fc epsilon RI) on rat basophilic leukemia cells using both single particle tracking and FPR. As in previous studies, our tracking results show that individual receptors may diffuse freely, or may exhibit restricted, time-dependent (anomalous) diffusion. Accordingly, we have analyzed FPR data by a new model to take this varied motion into account, and we show that the immobile fraction may be due to particles moving with the anomalous subdiffusion associated with restricted lateral mobility. Anomalous subdiffusion denotes random molecular motion in which the mean square displacements grow as a power law in time with a fractional positive exponent less than one. These findings call for a new model of cell membrane structure.

It has been proposed that the suppression of poleward flux within interpolar microtubule (ipMT) bundles of Drosophila embryonic spindles couples outward forces generated by a sliding filament mechanism to anaphase spindle elongation. Here, we (i) propose a molecular mechanism in which the bipolar kinesin KLP61F persistently slides dynamically unstable ipMTs outward, the MT depolymerase KLP10A acts at the poles to convert ipMT sliding to flux, and the chromokinesin KLP3A inhibits the depolymerase to suppress flux, thereby coupling ipMT sliding to spindle elongation; (ii) used KLP3A inhibitors to interfere with the coupling process, which revealed an inverse linear relation between the rates of flux and elongation, supporting the proposed mechanism and demonstrating that the suppression of flux controls both the rate and onset of spindle elongation; and (iii) developed a mathematical model using force balance and rate equations to describe how motors sliding the highly dynamic ipMTs apart can drive spindle elongation at a steady rate determined by the extent of suppression of flux.C hromosome segregation depends upon the action of the spindle, a protein machine that uses ensembles of kinesin and dynein motors plus microtubule (MT) dynamics to move chromatids polewards (anaphase A) and to elongate the spindle (anaphase B) (1). Anaphase B is driven in part by a bipolar kinesin-dependent sliding filament mechanism (2-9), with the extent of spindle elongation determined by MT polymerization in the overlap zone (2). Poleward flux, the movement of tubulin subunits from the MT plus ends facing the spindle equator to their minus ends at the poles (10-14), is proposed to constrain the length of metaphase spindles, with subsequent inhibition of depolymerization at the poles converting metaphase flux to anaphase spindle elongation (12,15,16).In support of this hypothesis, we observed that a suppression of poleward flux occurs at anaphase B onset: tubulin speckles within interpolar MTs (ipMTs) of Drosophila embryonic spindles fluxed toward the stationary poles of preanaphase B (herein meaning metaphase-anaphase A) spindles, but during anaphase B the speckles moved apart at the same rate as the poles (12). Here, we propose that three mitotic motors play critical roles in this process, based on previous studies (Fig. 1A). First, the bipolar kinesin KLP61F drives a sliding filament mechanism that underlies spindle elongation, because inhibiting KLP61F (in an Ncd-null mutant to circumvent the collapse of prometaphase spindles) inhibits anaphase B (9). Second, the kin I kinesin KLP10A depolymerizes ipMTs at the poles of preanaphase spindles, converting sliding to poleward flux; its inhibition leads to the premature suppression of flux and spindle elongation (14), suggesting that it is down-regulated at the onset of anaphase B. Finally, the chromokinesin KLP3A organizes ipMTs into bundles and is required for efficient anaphase spindle elongation (17).Here, we report experimental and theoretical results that provide a quantitative d...

In creating the mitotic spindle and the contractile ring, natural selection has engineered fascinating precision machines whose movements depend upon forces generated by ensembles of cytoskeletal proteins. These machines segregate chromosomes and divide the cell with high fidelity. Current research on the mechanisms and regulation of spindle morphogenesis, chromosome motility and cytokinesis emphasizes how ensembles of dynamic cytoskeletal polymers and multiple motors cooperate to generate the forces that guide the cell through mitosis and cytokinesis.

SUMMARYSensory cilia are assembled and maintained by kinesin-2-dependent intraflagellar transport (IFT). We investigated if two C. elegans α- and β-tubulin isotypes, identified via mutants that lack their cilium distal segments, are delivered to their assembly sites by IFT. Mutations in conserved residues in both tubulins destabilize distal singlet microtubules (MTs). One isotype, TBB-4, assembles into MTs at the tips of the axoneme core and distal segments, where the MT tip-tracker, EB1, is found, and localizes all along the cilium, whereas the other, TBA-5, concentrates in distal singlets. IFT assays, FRAP analysis and modeling suggest that the continual transport of sub-stoichiometric numbers of these tubulin subunits by the IFT machinery can maintain sensory cilia at their steady state length.

For more than 30 years, the fundamental goal in molecular motility has been to resolve force-generating motor protein structural changes. Although low-resolution structural studies have provided evidence for force-generating myosin rotations upon muscle activation, these studies did not resolve structural states of myosin in contracting muscle. Using electron paramagnetic resonance, we observed two distinct orientations of a spin label attached specifically to a single site on the light chain domain of myosin in relaxed scallop muscle fibers. The two probe orientations, separated by a 36°؎ 5°axial rotation, did not change upon muscle activation, but the distribution between them changed substantially, indicating that a fraction (17% ؎ 2%) of myosin heads undergoes a large (at least 30°) axial rotation of the myosin light chain domain upon force generation and muscle contraction. The resulting model helps explain why this observation has remained so elusive and provides insight into the mechanisms by which motor protein structural transitions drive molecular motility.Muscle contraction results from the relative sliding between actin and myosin filaments, and most models suggest that filament sliding is driven by a large structural change of actin-bound myosin heads, usually depicted as an axial rotation on the order of 45°(1-3). However, resolving distinct myosin structural states in muscle has been difficult, because active muscle contains a heterogeneous population of myosin heads, each independently cycling through different structural states. Changes in x-ray diffraction (4), birefringence (5), electron microscopy (6), and fluorescence (7) upon muscle contraction are consistent with myosin head motions, but evidence for distinct myosin orientational states has not been provided by these techniques, suggesting that either distinct orientations do not exist or these techniques have insufficient orientational resolution to detect them.Electron paramagnetic resonance (EPR) has the orientational resolution needed to detect multiple orientations of nitroxide spin labels, because each spin label orientation corresponds to a unique splitting between the three narrow spectral lines. Therefore, by covalently attaching a spin label to a specific site on myosin in muscle and orienting the muscle fiber in the magnetic field, myosin orientations with respect to the fiber axis can be determined. Multiple orientations of myosin in muscle, as well as the degree of disorder about those orientations, can be determined by resolving the spectrum of an oriented fiber into a sum of spectra with different splittings (8).Previous EPR studies revealed two structural states of the spin-labeled catalytic domain, the globular region of myosin that regulates actin-myosin crossbridges through ATP binding and hydrolysis. These structural states are characterized by a single orientation in the strong-binding biochemical states (high actin affinity) and microsecond dynamic disorder in the weak-binding biochemical states (low actin aff...

We used antibody microinjection and genetic manipulations to dissect the various roles of the homotetrameric kinesin-5, KLP61F, in astral, centrosome-controlled Drosophila embryo spindles and to test the hypothesis that it slides apart interpolar (ip) microtubules (MT), thereby controlling poleward flux and spindle length. In wild-type and Ncd null mutant embryos, anti-KLP61F dissociated the motor from spindles, producing a spatial gradient in the KLP61F content of different spindles, which was visible in KLP61F-GFP transgenic embryos. The resulting mitotic defects, supported by gene dosage experiments and time-lapse microscopy of living klp61f mutants, reveal that, after NEB, KLP61F drives persistent MT bundling and the outward sliding of antiparallel MTs, thereby contributing to several processes that all appear insensitive to cortical disruption. KLP61F activity contributes to the poleward flux of both ipMTs and kinetochore MTs and to the length of the metaphase spindle. KLP61F activity maintains the prometaphase spindle by antagonizing Ncd and another unknown force-generator and drives anaphase B, although the rate of spindle elongation is relatively insensitive to the motor's concentration. Finally, KLP61F activity contributes to normal chromosome congression, kinetochore spacing, and anaphase A rates. Thus, a KLP61F-driven sliding filament mechanism contributes to multiple aspects of mitosis in this system. INTRODUCTIONMitosis, the process by which identical copies of the replicated genome are distributed to the products of each cell division, involves a highly dynamic sequence of coordinated motility events, mediated by a bipolar protein machine, the mitotic spindle (Karsenti and Vernos, 2001;Mitchison and Salmon, 2001;Gadde and Heald, 2004;Wadsworth and Khodjakov, 2004;Mogilner et al., 2006;Brust-Mascher and Scholey, 2007;Walczak and Heald, 2008). These motility events are driven by molecular-scale forces generated by mitotic kinesins and dyneins, together with dynamic microtubules (MTs), whose activities are controlled by a network of regulatory proteins, e.g., mitotic kinases, phosphatases, and proteolytic enzymes (Sharp et al., 2000c; BettencourtDias et al., 2004;Maiato and Sunkel, 2004;Rogers et al., 2005;Goshima et al., 2007). Among these mitotic proteins, the kinesin-5 motor is thought to play a key role (Cottingham et al., 1999;Valentine et al., 2006a;Civelekoglu-Scholey and Scholey, 2007).Purified kinesin-5 is a slow, modestly processive, plusend-directed bipolar homotetramer capable of cross-linking adjacent MTs and sliding apart antiparallel MTs in motility assays (Sawin et al., 1992;Cole et al., 1994;Kashina et al., 1996a;Kapitein et al., 2005;Tao et al., 2006;Valentine et al., 2006b;Krzysiak et al., 2008;Van den Wildenberg et al., 2008). In yeast cells the homotetrameric structure of kinesin-5 appears to be essential for mitosis (Hildebrandt et al., 2006), and in Drosophila embryos KLP61F displays dynamic properties consistent with an association with spindle MTs (Cheerambathur et al., 2008) ...

The IgM Fc receptor (FcμR), originally cloned as “Fas-apoptosis inhibitory molecule (FAIM3/TOSO)” can function as a cell surface receptor for secreted IgM on a variety of cell types. We report that FcμR also is expressed in the trans-Golgi network of developing B cells, where it constrains IgM- but not IgD-BCR transport. In FcμR absence, IgM-BCR surface expression was increased, resulting in enhanced tonic BCR signaling. B cell-specific FcμR-deficiency enhanced spontaneous differentiation of B-1 cells, resulting in increases in natural IgM levels, and dysregulated B-2 cell homeostasis, causing spontaneous germinal center formation, increased serum autoantibody titers, and excessive B cell accumulation. Thus, FcμR/FAIM3 is a critical regulator of B cell biology by constraining IgM-BCR transport and cell surface expression.