@article {68634, title = {Distinct mobility patterns of BRCA2 molecules at DNA damage sites}, journal = {Nucleic Acids Res.}, year = {2023}, month = {2024 Jul 02}, pages = {gkae559}, abstract = {

BRCA2 is an essential tumor suppressor protein involved in promoting faithful repair of DNA lesions. The activity of BRCA2 needs to be tuned precisely to be active when and where it is needed. Here, we quantified the spatio-temporal dynamics of BRCA2 in living cells using aberration-corrected multifocal microscopy (acMFM). Using multicolor imaging to identify DNA damage sites, we were able to quantify its dynamic motion patterns in the nucleus and at DNA damage sites. While a large fraction of BRCA2 molecules localized near DNA damage sites appear immobile, an additional fraction of molecules exhibits subdiffusive motion, providing a potential mechanism to retain an increased number of molecules at DNA lesions. Super-resolution microscopy revealed inhomogeneous localization of BRCA2 relative to other DNA repair factors at sites of DNA damage. This suggests the presence of multiple nanoscale compartments in the chromatin surrounding the DNA lesion, which could play an important role in the contribution of BRCA2 to the regulation of the repair process.

}, doi = {10.1093/nar/gkae559 }, url = {https://doi.org/10.1093/nar/gkae559 }, author = {Maarten W. Paul and Jesse Aaron and Eric Wait and Romano M. van Genderen and Ihor Smal and Teng-Leong Chew and Roland Kanaar and Claire Wyman} } @article {68633, title = {Specialized actin nanoscale layers control focal adhesion turnover}, journal = {bioRxiv}, year = {2023}, month = {2023 Feb 15}, abstract = {

Focal adhesions (FAs) connect inner workings of the cell to the extracellular matrix to control cell adhesion, migration, and mechanosensing1,2. Previous studies demonstrated that FAs contain three vertical layers, which connect extracellular matrix to the cytoskeleton3,4,5. However, cellular processes rely on precisely-regulated FA turnover, but the molecular machineries that control FA assembly and disassembly have remained elusive. By using super-resolution iPALM microscopy, we identified two unprecedented nanoscale layers within FAs, specified by actin filaments bound to tropomyosin isoforms Tpm1.6 and Tpm3.2. The Tpm1.6-actin filaments beneath the previously identified {\textquoteleft}actin-regulatory layer{\textquoteright} are critical for adhesion maturation and controlled cell motility, whereas the Tpm3.2-actin filament layer towards the bottom of FA facilitates adhesion disassembly. Mechanistically, Tpm3.2 stabilizes KANK-family proteins at adhesions, and hence targets microtubule plus-ends to FAs to catalyse their disassembly. Loss of Tpm3.2 leads to disorganized microtubule network, abnormally stable FAs, and defects in tail retraction during cell migration. Thus, FAs are composed of at least three distinct actin filament layers, each having specific roles in coupling of adhesion to the cytoskeleton, or in controlling adhesion dynamics. In a broader context, these findings demonstrate how distinct actin filament populations can co-exist and perform specific functions within a defined cellular compartment.

}, doi = {10.1101/2023.02.15.528622}, url = {https://doi.org/10.1101/2023.02.15.528622}, author = {Reena Kumari and Katharina Ven and Megan Chastney and Johan Per{\"a}nen and Jesse Aaron and Leonardo Almeida-Souza and Elena Kremneva and Renaud Poincloux and Teng-Leong Chew and Peter W. Gunning and Johanna Ivaska and Pekka Lappalainen} } @article {68045, title = {Actin nano-architecture of phagocytic podosomes}, journal = {Nature Communications}, volume = {13}, year = {2022}, month = {2022 Jul 27}, pages = {4363}, abstract = {

Podosomes are actin-enriched adhesion structures important for multiple cellular processes, including migration, bone remodeling, and phagocytosis. Here, we characterized the structure and organization of phagocytic podosomes using interferometric photoactivated localization microscopy (iPALM), a super-resolution microscopy technique capable of 15-20 nm resolution, together with structured illumination microscopy (SIM) and localization-based superresolution microscopy. Phagocytic podosomes were observed during frustrated phagocytosis, a model in which cells attempt to engulf micro-patterned IgG antibodies. For circular patterns, this resulted in regular arrays of podosomes with well-defined geometry. Using persistent homology, we developed a pipeline for semi-automatic identification and measurement of podosome features. These studies revealed an "hourglass" shape of the podosome actin core, a protruding "knob" at the bottom of the core, and two actin networks extending from the core. Additionally, the distributions of paxillin, talin, myosin II, α-actinin, cortactin, and microtubules relative to actin were characterized.

}, doi = {10.1101/2022.05.04.490675}, author = {J. Cody Herron and Shiqiong Hu and Takashi Watanabe and Ana T. Nogueira and Bei Liu and Megan Kern and Jesse Aaron and Aaron Taylor and Michael Pablo and Teng-Leong Chew and Timothy C. Elston and Klaus M. Hahn} } @article {68105, title = {Single molecule microscopy to profile the effect of zinc status of transcription factor dynamics}, journal = {bioRxiv}, year = {2022}, month = {2022 May 11}, abstract = {

Transcription factors (TFs) are DNA binding proteins that control the expression of genes. The regulation of transcription is a complex process that involves binding of TFs to specific sequences, recruitment of cofactors and chromatin remodelers, assembly of the pre-initiation complex and ultimately the recruitment of RNA polymerase II. Increasing evidence suggests that TFs are highly dynamic and interact only transiently with DNA. Single molecule microscopy techniques are powerful approaches for visualizing and tracking individual TF molecules as they diffuse in the nucleus and interact with DNA. In this work, we employ multifocus microscopy and highly inclined and laminated optical sheet microscopy to track TF dynamics in response to perturbations in labile zinc inside cells. We sought to define whether zinc-dependent TFs sense changes in the labile zinc pool by determining whether their dynamics and DNA binding can be modulated by zinc. While it is widely appreciated that TFs need zinc to bind DNA, whether zinc occupancy and hence TF function are sensitive to\ changes\ in cellular zinc remain open questions. We utilized fluorescently tagged versions of the glucocorticoid receptor (GR), with two C4 zinc finger domains, and CCCTC-binding factor (CTCF), with eleven C2H2 zinc finger domains. We found that the biophysical dynamics of both TFs are susceptible to changes in zinc, but in subtly different ways. These results indicate that at least some transcription factors are sensitive to zinc dynamics, revealing a potential new layer of transcriptional regulation.

}, doi = {10.1101/2022.05.10.491421}, url = {https://doi.org/10.1101/2022.05.10.491421}, author = {Leah J. Damon and Jesse Aaron and Amy E. Palmer} } @article {65002, title = {Super resolution imaging of a distinct chromatin loop in human lymphoblastoid cells.}, journal = {bioRxiv}, year = {2019}, month = {2019 Apr 29}, abstract = {

The three-dimensional genome structure plays a fundamental role in gene regulation and cellular functions. Recent studies in genomics based on sequencing technologies inferred the very basic functional chromatin folding structures of the genome known as chromatin loops, the long-range chromatin interactions that are often mediated by protein factors. To visualize the looping structure of chromatin we applied super-resolution microscopy iPALM to image a specific chromatin loop in GM12878 cells. Totally, we have generated six images of the target chromatin region at the single molecule resolution. To infer the chromatin structures from the captured images, we modeled them as looping conformations using different computational algorithms and then evaluated the models by comparing with Hi-C data to examine the concordance. The results showed a good correlation between the imaging data and sequencing data, suggesting the visualization of higher-order chromatin structures for the very short genomic segments can be realized by microscopic imaging.

}, doi = {10.1101/621920}, author = {Jacqueline Jufen Zhu and Zofia Parteka and Byoungkoo Lee and Przemyslaw Szalaj and Ping Wang and Karolina Jodkowska and Jesse Aaron and Teng-Leong Chew and Dariusz Plewczynski and Yijun Ruan} } @article {64998, title = {Superresolution architecture of pluripotency guarding adhesions.}, journal = {bioRxiv}, year = {2018}, month = {2018 Sep 19}, abstract = {

Human pluripotent stem cells (hPSC) can generate almost all adult cell lineages. While it is clear that key transcriptional programmes are important elements for maintaining pluripotency, the equally essential requirement for cell adhesion to specific extracellular matrix components remains poorly defined. Our recent observation that hPSC colonies form unusually large {\textquotedblleft}cornerstone{\textquotedblright} focal adhesions (FA), distinct from parental somatic cells, that are lost following differentiation, emphasises the potential of these atypical FA as gatekeepers of pluripotency. Here, using nanopatterns, we further demonstrate that physical restriction of adhesion size, in hPSC colonies, is sufficient to trigger differentiation. Using superresolution two-colour interfero-metric photo-activated localization microscopy (iPALM), we examined the three-dimensional architecture of these cornerstone adhesions and report vertical lamination of FA proteins with three main structural peculiarities: 1) integrin β5 and talin are present at high density, at the edges of cornerstone FA, adjacent to a vertical kank-rich protein wall. 2) Vinculin localises higher than expected with respect to the substrata and displays a head-above-tail orientation, and 3) surprisingly, actin and α-actinin are present in two discrete layers, a previously undescribed localisation for these proteins. Finally, we report that depletion of kanks diminishes FA patterning, and actin organisation within the colony, indicating a key role for kanks in hPSC colony architecture.

}, doi = {10.1101/402305}, author = {Aki Stubb and Camilo Guzm{\'a}n and Elisa N{\"a}rv{\"a} and Jesse Aaron and Teng-Leong Chew and Markku Saari and Mitro Miihkinen and Guillaume Jacquemet and Johanna Ivaska} }