Microtubule cross-linking triggers the directional motility of kinesin-5

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

Arousal of Cancer-Associated Stroma: Overexpression of Palladin Activates Fibroblasts to Promote Tumor Invasion

Background: Cancer-associated fibroblasts, comprised of activated fibroblasts or myofibroblasts, are found in the stroma surrounding solid tumors. These myofibroblasts promote invasion and metastasis of cancer cells. Mechanisms regulating the activation of the fibroblasts and the initiation of invasive tumorigenesis are of great interest. Upregulation of the cytoskeletal protein, palladin, has been detected in the stromal myofibroblasts surrounding many solid cancers and in expression screens for genes involved in invasion. Using a pancreatic cancer model, we investigated the functional consequence of overexpression of exogenous palladin in normal fibroblasts in vitro and its effect on the early stages of tumor invasion. Principal Findings: Palladin expression in stromal fibroblasts occurs very early in tumorigenesis. In vivo, concordant expression of palladin and the myofibroblast marker, alpha smooth muscle actin (a-SMA), occurs early at the dysplastic stages in peri-tumoral stroma and progressively increases in pancreatic tumorigenesis. In vitro introduction of exogenous 90 kD palladin into normal human dermal fibroblasts (HDFs) induces activation of stromal fibroblasts into myofibroblasts as marked by induction of a-SMA and vimentin, and through the physical change of cell morphology. Moreover, palladin expression in the fibroblasts enhances cellular migration, invasion through the extracellular matrix, and creation of tunnels through which cancer cells can follow. The fibroblast invasion and creation of tunnels results from the development o

Solid phase synthesis and binding affinity of peptidyl transferase transition state mimics containing 2′-OH at P-site position A76

All living cells are dependent on ribosomes to catalyze the peptidyl transfer reaction, by which amino acids are assembled into proteins. The previously studied peptidyl transferase transition state analog CC-dA-phosphate-puromycin (CCdApPmn) has important differences from the transition state, yet current models of the ribosomal active site have been heavily influenced by the properties of this molecule. One significant difference is the substitution of deoxyadenosine for riboadenosine at A76, which mimics the 3′ end of a P-site tRNA. We have developed a solid phase synthetic approach to produce inhibitors that more closely match the transition state, including the critical P-site 2′-OH. Inclusion of the 2′-OH or an even bulkier OCH(3) group causes significant changes in binding affinity. We also investigated the effects of changing the A-site amino acid side chain from phenylalanine to alanine. These results indicate that the absence of the 2′-OH is likely to play a significant role in the binding and conformation of CCdApPmn in the ribosomal active site by eliminating steric clash between the 2′-OH and the tetrahedral phosphate oxygen. The conformation of the actual transition state must allow for the presence of the 2′-OH, and transition state mimics that include this critical hydroxyl group must bind in a different conformation from that seen in prior analog structures. These new inhibitors will provide valuable insights into the geometry and mechanism of the ribosomal active site

Data collected as described in , but in the presence of a mixture of 1 nM Eg5-GFP and 2 nM of unlabeled tetrameric Eg5

(A) Top kymograph shows sliding of a microtubule (MT) relative to a surface-attached microtubule. Below, the corresponding kymograph of Eg5-GFP shows directional runs between the overlapping microtubules (region marked with two red dotted lines). (B–F) Analysis of Eg5 motility during relative sliding. (B) Scatter plot of all pairs of short-term velocity and diffusion constant determined for a window of 15 s moving over the composite position-time trace of 94 Eg5 motors traced in the overlap zone of 11 microtubule pairs (2,335 points obtained from 2,349 s of total time). The horizontal dotted line indicates the average velocity of sliding microtubules (33 nm/s), and the vertical dotted line indicates the threshold used to discriminate slow and fast diffusion. (C and D) Similar analyses for Eg5 moving on individual microtubules at low ionic strength (C, 70 mM Pipes; ; 4,266 points) and high ionic strength (D, data pooled from 70 mM Pipes + 60 mM KCl and 70 mM Pipes + 80 mM KCl; 2,478 points). (E) Position-time traces. Black, fraction of the composite trace used for B. Green and red, sorted time points with a short-term diffusion constant; < 1,500 nm/s (green) and >1,500 nm/s (red). (F) Histograms of the short-term velocities as obtained from the time points in the green and red trace in E. The arrow indicates the average microtubule sliding velocity. (G) Graph summarizing Eg5 behavior under various conditions. Bar, 2 μm.<p><b>Copyright information:</b></p><p>Taken from "Microtubule cross-linking triggers the directional motility of kinesin-5"</p><p></p><p>The Journal of Cell Biology 2008;182(3):421-428.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2500128.</p><p></p

(A and B) Frames (A) and kymograph (B) from a time-lapse recording showing single molecules of Eg5-GFP (green) moving directionally along a microtubule (red) in the presence of 70 mM Pipes

(C and D) Frames (C) and kymograph (D) from a time-lapse recording showing single molecules of Eg5-GFP (green) diffusing along a microtubule (red) in the presence of 70 mM Pipes plus 40 mM KCl. (E–H) MD calculated from Eg5-GFP motility in the presence of ATP (black) and ADP (red) in 70 mM Pipes plus 0, 20, 40, or 60 mM KCl. Fits represent MD = . (I–M) MSD calculated from Eg5-GFP motility in the presence of ATP (black) and ADP (red) and at the indicated ionic strengths. Fits represent MD = and MD 2 + offset for the ATP data and MD 2 + offset for the ADP data. All numerical results are listed in . (N) Histogram of the duration of binding events for 0 mM KCl and for 60 mM KCl added. Lines are single exponential fits (exp[−t/t]) to the data (0 mM: t = 34 ± 3, = 212; 60 mM: t = 16 ± 2, = 119). Error bars represent SD. Bars, 1 μm.<p><b>Copyright information:</b></p><p>Taken from "Microtubule cross-linking triggers the directional motility of kinesin-5"</p><p></p><p>The Journal of Cell Biology 2008;182(3):421-428.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2500128.</p><p></p

(A) Kymograph of the displacement of 230 pM Eg5-513-GFP dimers versus time in 80 mM Pipes buffer with 0

2 mM ATP. A large majority of binding events last two frames or less (<p><b>Copyright information:</b></p><p>Taken from "Microtubule cross-linking triggers the directional motility of kinesin-5"</p><p></p><p>The Journal of Cell Biology 2008;182(3):421-428.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2500128.</p><p></p