@article {69656, title = {Leukocytes use endothelial membrane tunnels to extravasate the vasculature}, journal = {bioRxiv}, year = {2024}, month = {2024 Oct 30}, abstract = {

Upon inflammation, leukocytes extravasate through endothelial cells. When they extravasate in a paracellular manner, it is generally accepted that neighbouring endothelial cells physically disconnect to open cell-cell junctions, allowing leukocytes to cross. When carefully examining endothelial junctions, we found a partial membrane overlap of endothelial cells beyond VE-cadherin distribution. These overlaps are regulated by actin polymerization and, although marked by, do not require PECAM-1, nor VE-cadherin. Neutrophils prefer wider membrane overlaps as exit sites. Detailed 3D analysis of endothelial membrane dynamics during paracellular neutrophil transmigration in real-time, at high spatiotemporal resolution using resonant confocal and lattice light-sheet imaging, revealed that overlapping endothelial membranes form a tunnel during neutrophil transmigration. These tunnels are formed by the neutrophil lifting the membrane of the upper endothelial cell while indenting and crawling over the membrane of the underlying endothelial cell. Our work shows that endothelial cells do not simply retract upon passage of neutrophils but provide membrane tunnels, allowing neutrophils to extravasate. This discovery defines the 3D multicellular architecture in which the paracellular transmigration of neutrophils occurs.

}, doi = {10.1101/2024.10.28.620560}, url = {https://doi.org/10.1101/2024.10.28.620560}, author = {van der Meer, Werner and van Steen, Abraham C.I. and Mahlandt, Eike and Rolas, Loic and Wang, Haitao and Arts, Janine and Kempers, Lanette and Gr{\"o}nloh, Max and Schoon, Rianne M. and Driessen, Amber and van Rijssel, Jos and Klaassen, Ingeborg and Schlingemann, Reinier O. and Manavski, Yosif and Hoogenboezem, Mark and Boon, Reinier A. and Khuon, Satya and Wait, Eric and Heddleston, John and Chew, Teng-Leong and Nolte, Martijn A and Nourshargh, Sussan and Goedhart, Joachim and van Buul, Jaap D} } @article {66021, title = {The level of oncogenic Ras determines the malignant transformation of Lkb1 mutant tissue in vivo.}, journal = {Communications Biology}, volume = {4}, year = {2021}, month = {2021 Jan 29}, pages = {142}, abstract = {

The genetic and metabolic heterogeneity of RAS-driven cancers has confounded therapeutic strategies in the clinic. To address this, rapid and genetically tractable animal models are needed that recapitulate the heterogeneity of RAS-driven cancers in vivo. Here, we generate a Drosophila melanogaster model of Ras/Lkb1 mutant carcinoma. We show that low-level expression of oncogenic Ras (Ras) promotes the survival of Lkb1 mutant tissue, but results in autonomous cell cycle arrest and non-autonomous overgrowth of wild-type tissue. In contrast, high-level expression of oncogenic Ras (Ras) transforms Lkb1 mutant tissue resulting in lethal malignant tumors. Using simultaneous multiview light-sheet microcopy, we have characterized invasion phenotypes of Ras/Lkb1 tumors in living larvae. Our molecular analysis reveals sustained activation of the AMPK pathway in malignant Ras/Lkb1 tumors, and demonstrate the genetic and pharmacologic dependence of these tumors on CaMK-activated Ampk. We further show that LKB1 mutant human lung adenocarcinoma patients with high levels of oncogenic KRAS exhibit worse overall survival and increased AMPK activation. Our results suggest that high levels of oncogenic KRAS is a driving event in the malignant transformation of LKB1 mutant tissue, and uncovers a vulnerability that may be used to target this aggressive genetic subset of RAS-driven tumors.

}, issn = {2399-3642}, doi = {10.1038/s42003-021-01663-8}, author = {Rackley, Briana and Seong, Chang-Soo and Kiely, Evan and Parker, Rebecca E and Rupji, Manali and Dwivedi, Bhakti and Heddleston, John M and Giang, William and Anthony, Neil and Chew, Teng-Leong and Gilbert-Ross, Melissa} } @article {48219, title = {Long-range self-organization of cytoskeletal myosin II filament stacks.}, journal = {Nature Cell Biology}, volume = {19}, year = {2017}, month = {2017 Jan 23}, pages = {133-41}, abstract = {

Although myosin II filaments are known to exist in non-muscle cells, their dynamics and organization are incompletely understood. Here, we combined structured illumination microscopy with pharmacological and genetic perturbations, to study the process of actomyosin cytoskeleton self-organization into arcs and stress fibres. A striking feature of the myosin II filament organization was their {\textquoteright}registered{\textquoteright} alignment into stacks, spanning up to several micrometres in the direction orthogonal to the parallel actin bundles. While turnover of individual myosin II filaments was fast (characteristic half-life time 60 s) and independent of actin filament turnover, the process of stack formation lasted a longer time (in the range of several minutes) and required myosin II contractility, as well as actin filament assembly/disassembly and crosslinking (dependent on formin Fmnl3, cofilin1 and α-actinin-4). Furthermore, myosin filament stack formation involved long-range movements of individual myosin filaments towards each other suggesting the existence of attractive forces between myosin II filaments. These forces, possibly transmitted via mechanical deformations of the intervening actin filament network, may in turn remodel the actomyosin cytoskeleton and drive its self-organization.

}, issn = {1476-4679}, doi = {10.1038/ncb3466}, author = {Hu, Shiqiong and Dasbiswas, Kinjal and Guo, Zhenhuan and Tee, Yee-Han and Thiagarajan, Visalatchi and Hersen, Pascal and Chew, Teng-Leong and Safran, Samuel A and Zaidel-Bar, Ronen and Bershadsky, Alexander D} } @article {47937, title = {Light sheet microscopes: Novel imaging toolbox for visualizing life{\textquoteright}s processes.}, journal = {The International Journal of Biochemistry \& Cell Biology}, year = {2016}, month = {2016 Oct 7}, abstract = {

Capturing dynamic processes in live samples is a nontrivial task in biological imaging. Although fluorescence provides high specificity and contrast compared to other light microscopy techniques, the photophysical principles of this method can have a harmful effect on the sample. Current advances in light sheet microscopy have created a novel imaging toolbox that allows for rapid acquisition of high-resolution fluorescent images with minimal perturbation of the processes of interest. Each unique design has its own advantages and limitations. In this review, we describe several cutting edge light sheet microscopes and their optimal applications.

}, issn = {1878-5875}, doi = {10.1016/j.biocel.2016.10.002}, author = {Heddleston, John M and Chew, Teng-Leong} }