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.Molecular and Cellular Biolog

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.National Institutes of Health (U.S.) (NIH GM102486)National Science Foundation (U.S.) (NSF CMMI 0954302, NSF CMMI 0927945, and NSF CTS-0505929

Actomyosin Pulls to Advance the Nucleus in a Migrating Tissue Cell

AbstractThe 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

Direct force probe reveals the mechanics of nuclear homeostasis in the mammalian cell

How cells maintain nuclear shape and position against various intracellular and extracellular forces is not well understood, although defects in nuclear mechanical homeostasis are associated with a variety of human diseases. We estimated the force required to displace and deform the nucleus in adherent living cells with a technique to locally pull the nuclear surface. A minimum pulling force of a few nanonewtons--far greater than typical intracellular motor forces--was required to significantly displace and deform the nucleus. Upon force removal, the original shape and position were restored quickly within a few seconds. This stiff, elastic response required the presence of vimentin, lamin A/C, and SUN (Sad1p, UNC-84)-domain protein linkages, but not F-actin or microtubules. Although F-actin and microtubules are known to exert mechanical forces on the nuclear surface through molecular motor activity, we conclude that the intermediate filament networks maintain nuclear mechanical homeostasis against localized forces

The mammalian LINC complex regulates genome transcriptional responses to substrate rigidity

Mechanical integration of the nucleus with the extracellular matrix (ECM) is established by linkage between the cytoskeleton and the nucleus. This integration is hypothesized to mediate sensing of ECM rigidity, but parsing the function of nucleus-cytoskeleton linkage from other mechanisms has remained a central challenge. Here we took advantage of the fact that the LINC (linker of nucleoskeleton and cytoskeleton) complex is a known molecular linker of the nucleus to the cytoskeleton, and asked how it regulates the sensitivity of genome-wide transcription to substratum rigidity. We show that gene mechanosensitivity is preserved after LINC disruption, but reversed in direction. Combined with myosin inhibition studies, we identify genes that depend on nuclear tension for their regulation. We also show that LINC disruption does not attenuate nuclear shape sensitivity to substrate rigidity. Our results show for the first time that the LINC complex facilitates mechano-regulation of expression across the genome

FRAP Analysis: Accounting for Bleaching during Image Capture

<div><p>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.</p> </div

Solutions to Eq. 9a and 9b that account for the presence of an actual immobile fraction.

<p>(A) Observed recovery curves with  = 0.4,  = 0.3 (immobile fraction),  = 0.2, <<1 (i.e. negligible photobleaching of the cytoplasmic molecules such that ) and  = 1 (*), 0.5 (<b>◊</b>), 0.25 (<b>□</b>), 0.1 (<b>○</b>) (from top to bottom). (B) Illustration of behavior of mobile (dashed curve) and immobile fractions (dotted curve) during recovery for  = 1 (* indicates total intensity). The immobile fraction can be seen to decay due to bleaching during image capture, resulting in a decrease in the total fluorescent intensity. (C) Effect of the immobile fraction on the observed recovery curves.  = 0.4,  = 0.2, <<1,  = 1 and  = 0.2 (*), 0.4 (<b>◊</b>), 0.6 (<b>□</b>), 0.8 (<b>○</b>) (from top to bottom). Pronounced transients are observed in the recovery. (D) Effect of the bleaching function on recovery. Observed recovery curves with  = 0.4,  = 0.3, <<1,  = 1, and  = 10⁻⁶ (*), 0.2 (<b>◊</b>), 0.46 (<b>□</b>), 1.1 (<b>○</b>) (from top to bottom). Solid triangles at in all figures indicate the normalized initial intensity before the photobleaching.</p

Solutions to Eq. 6 showing how bleaching during image capture can give the erroneous impression of an ‘immobile’ fraction.

<p>Recovery curves are shown with  = 0.4,  = 0.2,  = 1 (*), 0.5 (◊), 0.25 (□) and 0.1 (○) (from top to bottom). For plotting purposes, is assumed to be /10. The solid triangle at indicates the normalized initial intensity before photobleaching.</p

Effect of bleaching due to image capture on measured fluorescence.

<p>(A) shows calculations of Eq (2) for and (as the actual concentration is not measured in experiments, the value of is not relevant). The dotted lines indicate the actual dynamics including the decay of the fluorescence due to bleaching during image capture. * indicates the averaged concentration in an image. B) Dotted curves are normalized concentrations calculated from Eq (3). Normalizing average concentrations with the concentration in the first image yields similar dynamics, except the effect of averaging on the measured concentration is cancelled (this is discussed more in the text) such that the normalized average fluorescence is equal to . (C) Hypothetical effect of bleaching during image capture on FRAP recovery. The dotted curve is the actual dynamics consisting of (unobserved) recovery interspersed by bleaching during image capture, * indicates measured intensity. The solid triangle at indicates the normalized initial intensity before photobleaching.</p