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1.  Force fluctuations in three-dimensional suspended fibroblasts 
Cells are sensitive to mechanical cues from their environment and at the same time generate and transmit forces to their surroundings. To test quantitatively forces generated by cells not attached to a substrate, we used a dual optical trap to suspend 3T3 fibroblasts between two fibronectin-coated beads. In this simple geometry, we measured both the cells' elastic properties and the force fluctuations they generate with high bandwidth. Cell stiffness decreased substantially with both myosin inhibition by blebbistatin and serum-starvation, but not with microtubule depolymerization by nocodazole. We show that cortical forces generated by non-muscle myosin II deform the cell from its rounded shape in the frequency regime from 0.1 to 10 Hz. The amplitudes of these forces were strongly reduced by blebbistatin and serum starvation, but were unaffected by depolymerization of microtubules. Force fluctuations show a spectrum that is characteristic for an elastic network activated by random sustained stresses with abrupt transitions.
PMCID: PMC4275901  PMID: 25533089
cell mechanics; optical trap; cell cortex; non-muscle myosin II; active matter
2.  A surprising twist 
eLife  2014;3:e02715.
X-ray crystallography has revealed an unusual structural element in kinesin-5 motor proteins.
PMCID: PMC3978768  PMID: 24714499
mitosis; kinesin-5; microtubule; motor proteins; coiled-coil; x-ray structure; D. melanogaster
3.  Regulation of bi-directional movement of single kinesin-5 Cin8 molecules 
Bioarchitecture  2012;2(2):70-74.
Kinesin-5 mechanoenzymes drive mitotic spindle dynamics as slow, processive microtubule (MT)-plus-end directed motors. Surprisingly, the Saccharomyces cerevisiae kinesin-5 Cin8 was recently found to be bi-directional: it can move processively in both directions on MTs. Two hypotheses have been suggested for the mechanism of the directionality switch: (1) single molecules of Cin8 are intrinsically minus-end directed, but mechanical coupling between two or more motors triggers the switch; (2) a single motor can switch direction, and “cargo binding” i.e., binding between two MTs triggers the switch to plus-end motility. Single-molecule fluorescence data we published recently, and augment here, favor hypothesis (2). In low-ionic-strength conditions, single molecules of Cin8 move in both minus- and plus-end directions. Fluorescence photo bleaching data rule out aggregation of Cin8 while they move in the plus and in the minus direction. The evidence thus points toward cargo regulation of directionality, which is likely to be related to cargo regulation in other kinesins. The molecular mechanisms of this regulation, however, remain to be elucidated.
PMCID: PMC3383724  PMID: 22754632
Saccharomyces cerevisiae Cin8; kinesin directionality; kinesin-5; microtubules; mitosis
4.  Microtubule cross-linking triggers the directional motility of kinesin-5 
The Journal of Cell Biology  2008;182(3):421-428.
Although assembly of the mitotic spindle is known to be a precisely controlled process, regulation of the key motor proteins involved remains poorly understood. In eukaryotes, homotetrameric kinesin-5 motors are required for bipolar spindle formation. Eg5, the vertebrate kinesin-5, has two modes of motion: an adenosine triphosphate (ATP)–dependent directional mode and a diffusive mode that does not require ATP hydrolysis. We use single-molecule experiments to examine how the switching between these modes is controlled. We find that Eg5 diffuses along individual microtubules without detectable directional bias at close to physiological ionic strength. Eg5's motility becomes directional when bound between two microtubules. Such activation through binding cargo, which, for Eg5, is a second microtubule, is analogous to known mechanisms for other kinesins. In the spindle, this might allow Eg5 to diffuse on single microtubules without hydrolyzing ATP until the motor is activated by binding to another microtubule. This mechanism would increase energy and filament cross-linking efficiency.
PMCID: PMC2500128  PMID: 18678707
5.  High-resolution microrheology in the pericellular matrix of prostate cancer cells 
Many cells express a membrane-coupled external mechanical layer, the pericellular matrix (PCM), which often contains long-chain polymers. Its role and properties are not entirely known, but its functions are believed to include physical protection, mechanosensing, chemical signalling or lubrication. The viscoelastic response of the PCM, with polysaccharides as the main structural components, is therefore crucial for the understanding of its function. We have here applied microrheology, based on optically trapped micrometre-sized colloids, to the PCM of cultured PC3 prostate cancer cells. This technology allowed us to measure the extremely soft response of the PCM, with approximately 1 µm height resolution. Exogenously added aggrecan, a hyaluronan-binding proteoglycan, caused a remarkable increase in thickness of the viscoelastic layer and also triggered filopodia-like protrusions. The viscoelastic response of the PCM, however, did not change significantly.
PMCID: PMC3385749  PMID: 22319113
hyaluronan; optical tweezers; prostate cancer cells; elastic shear modulus
6.  Endoplasmic Reticulum Sorting and Kinesin-1 Command the Targeting of Axonal GABAB Receptors 
PLoS ONE  2012;7(8):e44168.
In neuronal cells the intracellular trafficking machinery controls the availability of neurotransmitter receptors at the plasma membrane, which is a critical determinant of synaptic strength. Metabotropic γ amino-butyric acid (GABA) type B receptors (GABABRs) are neurotransmitter receptors that modulate synaptic transmission by mediating the slow and prolonged responses to GABA. GABABRs are obligatory heteromers constituted by two subunits, GABABR1 and GABABR2. GABABR1a and GABABR1b are the most abundant subunit variants. GABABR1b is located in the somatodendritic domain whereas GABABR1a is additionally targeted to the axon. Sushi domains located at the N-terminus of GABABR1a constitute the only difference between both variants and are necessary and sufficient for axonal targeting. The precise targeting machinery and the organelles involved in sorting and transport have not been described. Here we demonstrate that GABABRs require the Golgi apparatus for plasma membrane delivery but that axonal sorting and targeting of GABABR1a operate in a pre-Golgi compartment. In the axon GABABR1a subunits are enriched in the endoplasmic reticulum (ER), and their dynamic behavior and colocalization with other secretory organelles like the ER-to-Golgi intermediate compartment (ERGIC) suggest that they employ a local secretory route. The transport of axonal GABABR1a is microtubule-dependent and kinesin-1, a molecular motor of the kinesin family, determines axonal localization. Considering that progression of GABABRs through the secretory pathway is regulated by an ER retention motif our data contribute to understand the role of the axonal ER in non-canonical sorting and targeting of neurotransmitter receptors.
PMCID: PMC3428321  PMID: 22952914
7.  The Homotetrameric Kinesin-5, KLP61F, Preferentially Crosslinks Microtubules into Antiparallel Orientations 
Current biology : CB  2008;18(23):1860-1864.
The segregation of the genetic material during mitosis is coordinated by the mitotic spindle, whose mechanism of action depends upon the polarity patterns of its constituent microtubules (MTs)[1, 2]. Homotetrameric mitotic kinesin-5 motors are capable of crosslinking and sliding adjacent spindle MTs [3-11], but it is unknown if they, or other motors, contribute to the establishment of these MT polarity patterns. Here we explored if the Drosophila embryo kinesin-5, KLP61F, which is thought to crosslink both parallel and anti-parallel MTs [7, 12], displays a preference for the parallel or anti-parallel orientation of MTs. In motility assays, KLP61F was observed to crosslink and slide adjacent MTs, as predicted. Remarkably, KLP61F displayed a three-fold higher preference for crosslinking MTs in the antiparallel, relative to the parallel orientation. This polarity preference was observed in the presence of ADP or in ATP plus AMPPNP, but not in AMPPNP alone, which induces instantaneous rigor binding. Also, a purified motorless tetramer containing the C-terminal tail domains displayed an antiparallel orientation preference, confirming that motor activity is not required. The results suggest that, during the morphogenesis of the Drosophila embryo mitotic spindle, the crosslinking and sliding activities of KLP61F could facilitate the gradual accumulation of KLP61F within antiparallel interpolar (ip) MTs at the equator, where the motor could then generate force to drive poleward flux and pole-pole separation.
PMCID: PMC2657206  PMID: 19062285
8.  Load-dependent release limits the processive stepping of the tetrameric Eg5 motor 
European Biophysics Journal   2007;36(6):675-681.
Tetrameric motor proteins of the Kinesin-5 family are essential for eukaryotic cell division. The microscopic mechanism by which Eg5, the vertebrate Kinesin-5, drives bipolar mitotic spindle formation remains unknown. Here we show in optical trapping experiments that full-length Eg5 moves processively and stepwise along microtubule bundles. Interestingly, the force produced by individual Eg5 motors typically reached only ∼2 pN, one-third of the stall force of Kinesin-1. Eg5 typically detached from microtubules before stalling. This behavior may reflect a regulatory mechanism important for the role of Eg5 in the mitotic spindle.
PMCID: PMC1914257  PMID: 17333163

Results 1-8 (8)