Actomyosin pulls to advance the nucleus in a migrating tissue cell.

The cytoskeletal forces involved in translocating the nucleus in a migrating tissue cell remain unresolved. Previous studies have variously implicated actomyosin-generated pushing or pulling forces on the nucleus, as well as pulling by nucleus-bound microtubule motors. We found that the nucleus in an isolated migrating cell can move forward without any trailing-edge detachment. When a new lamellipodium was triggered with photoactivation of Rac1, the nucleus moved toward the new lamellipodium. This forward motion required both nuclear-cytoskeletal linkages and myosin activity. Apical or basal actomyosin bundles were found not to translate with the nucleus. Although microtubules dampen fluctuations in nuclear position, they are not required for forward translocation of the nucleus during cell migration. Trailing-edge detachment and pulling with a microneedle produced motion and deformation of the nucleus suggestive of a mechanical coupling between the nucleus and the trailing edge. Significantly, decoupling the nucleus from the cytoskeleton with KASH overexpression greatly decreased the frequency of trailing-edge detachment. Collectively, these results explain how the nucleus is moved in a crawling fibroblast and raise the possibility that forces could be transmitted from the front to the back of the cell through the nucleus.

FLUCTUATING MOTOR FORCES BEND GROWING MICROTUBULES.

Despite their rigidity, microtubules in living cells bend significantly during polymerization resulting in greater curvature than can be explained by thermal forces alone. However, the source of the non-thermal forces that bend growing microtubules remains obscure. We analyzed the motion of microtubule tips in NIH-3T3 fibroblasts expressing EGFP-EB1, a fluorescent +TIP protein that specifically binds to the growing ends of microtubules. We found that dynein inhibition significantly reduced the deviation of the growing tip from its initial trajectory. Inhibiting myosin modestly reduced tip fluctuations, while simultaneous myosin and dynein inhibition caused no further decrease in fluctuations compared to dynein inhibition alone. Our results can be interpreted with a model in which dynein linkages play a key role in generating and transmitting fluctuating forces that bend growing microtubules.

Modulation of Nuclear Shape by Substrate Rigidity.

The nucleus is mechanically coupled to the three cytoskeletal elements in the cell via linkages maintained by the LINC complex (for Linker of Nucleoskeleton to Cyto-skeleton). It has been shown that mechanical forces from the extracellular matrix (ECM) can be transmitted through the cytoskeleton to the nuclear surface. Here we quantified nuclear shape in NIH 3T3 fibroblasts on polyacrylamide gels with a controlled degree of cross-linking. On soft substrates with a Young's modulus of 0.4 kPa, the nucleus appeared rounded in its vertical cross-section, while on stiff substrates (308 kPa), the nucleus appears more flattened. Over-expression of dominant negative Klarsicht ANC-1 Syne Homology (KASH) domains, which disrupts the LINC complex, eliminated the sensitivity of nuclear shape to substrate rigidity; myosin inhibition had similar effects. GFP-KASH4 over-expression altered the rigidity dependence of cell motility and cell spreading. Taken together, our results suggest that nuclear shape is modulated by substrate rigidity-induced changes in actomyosin tension, and that a mechanically integrated nucleus-cytoskeleton is required for rigidity sensing. These results are significant because they suggest that substrate rigidity can potentially control nuclear function and hence cell function.

Cytoplasmic dynein: tension generation on microtubules and the nucleus.

Cytoplasmic dynein is a microtubule dependent motor protein that is central to vesicle transport, cell division and organelle positioning. Recent studies suggest that dynein can generate significant pulling forces on intracellular structures as it motors along microtubules. In this review, we discuss how dynein-generated pulling forces position the nucleus and the centrosome.

FRAP analysis: accounting for bleaching during image capture.

The analysis of Fluorescence Recovery After Photobleaching (FRAP) experiments involves mathematical modeling of the fluorescence recovery process. An important feature of FRAP experiments that tends to be ignored in the modeling is that there can be a significant loss of fluorescence due to bleaching during image capture. In this paper, we explicitly include the effects of bleaching during image capture in the model for the recovery process, instead of correcting for the effects of bleaching using reference measurements. Using experimental examples, we demonstrate the usefulness of such an approach in FRAP analysis.