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.
H3K14ac (acetylation of lysine 14 of histone H3) is one of the most important epigentic modifications. Aberrant changes in H3K14ac have been associated with various diseases, including cancers and neurological disorders. Tools that enable detection and quantification of H3K14ac levels in cell extracts and in situ are thus of critical importance to reveal its role in various biological processes. Current detection techniques of specific histone modifications, however, are constrained by tedious sample pretreatments, lack of quantitative accuracy, and reliance on high quality antibodies. To address this issue, we engineered recombinant sensors that are suitable for probing histone acetylation levels using various biological samples. The protein sensor contains recongition domain(s) with sequences derived from the bromodomain of human polybromo-1 (PB1), a natural H3K14ac reader domain. Various sensor designs were tested using nuclear extracts and live cells. The sensor containing dimeric repeats of bromodomain was found most effective in quantifying H3K14ac level in both in vitro and in situ assays. The sensor has a linear detection range of 0.5-50 nM when mixed with nuclear extracts. The sensor colocalizes with H3K14ac antibodies in situ when transfected into human embryonic kidney 293T (HEK293T) cells and is thus capable of providing spatial details of histone modification within the nucleus. Corrected nuclear fluorescence intensity was used to quantify the modification level in situ and found to correlate well with our in vitro assays. Our sensor offers a novel tool to characterize the histone modification level using nuclear extracts and probe histone modification change in live cells.
H3K9me3 (methylation of lysine 9
of histone H3) is an epigenetic
modification that acts as a repressor mark. Several diseases, including
cancers and neurological disorders, have been associated with aberrant
changes in H3K9me3 levels. Different tools have been developed to
enable detection and quantification of H3K9me3 levels in cells. Most
techniques, however, lack live cell compatibility. To address this
concern, we have engineered recombinant protein sensors for probing
H3K9me3 in situ. A heterodimeric sensor containing a chromodomain
and chromo shadow domain from HP1a was found to be optimal in recognizing
H3K9me3 and exhibited similar spatial resolution to commercial antibodies.
Our sensor offers similar quantitative accuracy in characterizing
changes in H3K9me3 compared to antibodies but claims single cell resolution.
The sensor was applied to evaluate changes in H3K9me3 responding to
environmental chemical atrazine (ATZ). ATZ was found to result in
significant reductions in H3K9me3 levels after 24 h of exposure. Its
impact on the distribution of H3K9me3 among cell populations was also
assessed and found to be distinctive. We foresee the application of
our sensors in multiple toxicity and drug-screening applications.
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