@article {69801, title = {De-risking transformative microscopy technologies for broad adoption.}, journal = {J Microsc}, year = {2025}, month = {2025 Feb 27}, abstract = {

The past 20 years have seen a paradigm-shifting explosion of new optical microscopy technologies aimed at uncovering fundamental biological insights. Yet only a small portion {\textquoteright}cross the finish line{\textquoteright} into wide adoption by the life science community. We contend that this is not primarily due to a lack of technical prowess or utility. Rather, many risks can conspire to derail the adoption of potentially disruptive technologies. One way to address these challenges is to de-risk paradigm-shifting inventions within open-access technology incubators. Here we detail the framework needed to shepherd innovative microscopy techniques through the often-treacherous adoption landscape to enable transformative scientific output.

}, issn = {1365-2818}, doi = {10.1111/jmi.13400}, author = {Aaron, Jesse and Chew, Teng-Leong} } @article {69442, title = {Coumarin as a general switching auxiliary to prepare photochromic and spontaneously blinking fluorophores}, journal = {bioRxiv}, year = {2024}, month = {2024 May 12}, abstract = {

Single-molecule localization microscopy (SMLM) uses activatable or switchable fluorophores to create non-diffraction limited maps of molecular location in biological samples. Despite the utility of this imaging technique, the portfolio of appropriate labels for SMLM remains limited. Here, we describe a general strategy for the construction of {\textquotedblleft}glitter bomb{\textquotedblright} labels by simply combining rhodamine and coumarin dyes though an amide bond. Condensation of the ortho-carboxyl group on the pendant phenyl ring of rhodamine dyes with a 7-aminocoumarin yields photochromic or spontaneously blinking fluorophores depending on the parent rhodamine structure. We apply this strategy to prepare labels useful super-resolution experiments in fixed cells using different attachment techniques. This general glitter bomb strategy should lead to improved labels for SMLM, ultimately enabling the creation of detailed molecular maps in biological samples.

}, doi = {10.1101/2024.05.12.593749}, url = {https://www.biorxiv.org/content/early/2024/05/12/2024.05.12.593749}, author = {Jradi, Fadi M. and English, Brian P. and Brown, Timothy A. and Aaron, Jesse and Khuon, Satya and Galbraith, James A. and Galbraith, Catherine G. and Lavis, Luke D.} } @article {69320, title = {Focal adhesions contain three specialized actin nanoscale layers}, journal = {Nature Communications}, volume = {15}, year = {2024}, month = {2024 Mar 21}, abstract = {

Focal adhesions (FAs) connect inner workings of cell to the extracellular matrix to control cell adhesion, migration and mechanosensing. Previous studies demonstrated that FAs contain three vertical layers, which connect extracellular matrix to the cytoskeleton. By using super-resolution iPALM microscopy, we identify two additional 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 α-actinin cross-linked actin filaments, appear critical for adhesion maturation and controlled cell motility, whereas the adjacent Tpm3.2-actin filament layer beneath seems to facilitate adhesion disassembly. Mechanistically, Tpm3.2 stabilizes ACF-7/MACF1 and KANK-family proteins at adhesions, and hence targets microtubule plus-ends to FAs to catalyse their disassembly. Tpm3.2 depletion leads to disorganized microtubule network, abnormally stable FAs, and defects in tail retraction during migration. Thus, FAs are composed of distinct actin filament layers, and each may have specific roles in coupling adhesions to the cytoskeleton, or in controlling adhesion dynamics.

}, doi = {10.1038/s41467-024-46868-7}, url = {https://www.nature.com/articles/s41467-024-46868-7https://www.nature.com/articles/s41467-024-46868-7.pdfhttps://www.nature.com/articles/s41467-024-46868-7.pdfhttps://www.nature.com/articles/s41467-024-46868-7}, author = {Kumari, Reena and Ven, Katharina and Chastney, Megan and Kokate, Shrikant B. and Per{\"a}nen, Johan and Aaron, Jesse and Kogan, Konstantin and Almeida-Souza, Leonardo and Kremneva, Elena and Poincloux, Renaud and Chew, Teng-Leong and Gunning, Peter W. and Ivaska, Johanna and Lappalainen, Pekka} } @article {69461, title = {Four-dimensional quantitative analysis of cell plate development in Arabidopsis using lattice light sheet microscopy identifies robust transition points between growth phases}, journal = {J Exp Bot}, volume = {75}, year = {2024}, month = {2024 May 20}, pages = {2829-2847}, abstract = {

Cell plate formation during cytokinesis entails multiple stages occurring concurrently and requiring orchestrated vesicle delivery, membrane remodelling, and timely deposition of polysaccharides, such as callose. Understanding such a dynamic process requires dissection in time and space; this has been a major hurdle in studying cytokinesis. Using lattice light sheet microscopy (LLSM), we studied cell plate development in four dimensions, through the behavior of yellow fluorescent protein (YFP)-tagged cytokinesis-specific GTPase RABA2a vesicles. We monitored the entire duration of cell plate development, from its first emergence, with the aid of YFP-RABA2a, in both the presence and absence of cytokinetic callose. By developing a robust cytokinetic vesicle volume analysis pipeline, we identified distinct behavioral patterns, allowing the identification of three easily trackable cell plate developmental phases. Notably, the phase transition between phase I and phase II is striking, indicating a switch from membrane accumulation to the recycling of excess membrane material. We interrogated the role of callose using pharmacological inhibition with LLSM and electron microscopy. Loss of callose inhibited the phase transitions, establishing the critical role and timing of the polysaccharide deposition in cell plate expansion and maturation. This study exemplifies the power of combining LLSM with quantitative analysis to decode and untangle such a complex process.

}, keywords = {Arabidopsis, Cytokinesis, Glucans, Microscopy}, issn = {1460-2431}, doi = {10.1093/jxb/erae091}, author = {Sinclair, Rosalie and Wang, Minmin and Jawaid, Muhammad Zaki and Longkumer, Toshisangba and Aaron, Jesse and Rossetti, Blair and Wait, Eric and McDonald, Kent and Cox, Daniel and Heddleston, John and Wilkop, Thomas and Drakakaki, Georgia} } @article {69252, title = {Four-dimensional quantitative analysis of cell plate development using lattice light sheet microscopy identifies robust transition points between growth phases.}, journal = {Journal of Experimental Botany}, year = {2024}, month = {2024 Mar 4}, abstract = {

Cell plate formation during cytokinesis entails multiple stages occurring concurrently and requiring orchestrated vesicle delivery, membrane remodeling, and timely polysaccharide deposition, such as callose. Understanding such a dynamic process requires dissection in time and space; this has been a major hurdle in studying cytokinesis. Using lattice light sheet microscopy (LLSM) we studied cell plate development in four dimensions, through the behavior of the cytokinesis specific GTPase YFP-RABA2a vesicles. We monitored the entire length of cell plate development, from its first emergence, with the aid of YFP-RABA2a, both in the presence and absence of cytokinetic callose. By developing a robust cytokinetic vesicle volume analysis pipeline, we identified distinct behavioral patterns, allowing the identification of three easily trackable, cell plate developmental phases. Notably, the phase transition between phase I and phase II is striking, indicating a switch from membrane accumulation to the recycling of excess membrane material. We interrogated the role of callose using pharmacological inhibition with LLSM and electron microscopy. Loss of callose inhibited the phase transitions, establishing the critical role and timing of the polysaccharide deposition in cell plate expansion and maturation. This study exemplifies the power of combining LLSM with quantitative analysis to decode and untangle such a complex process.

}, issn = {1460-2431}, doi = {10.1093/jxb/erae091}, author = {Sinclair, Rosalie and Wang, Minmin and Jawaid, Muhammad Zaki and Longkumer, Toshisangba and Aaron, Jesse and Rossetti, Blair and Wait, Eric and McDonald, Kent and Cox, Daniel and Heddleston, John and Wilkop, Thomas and Drakakaki, Georgia} } @article {68787, title = {Autophagy receptor NDP52 alters DNA conformation to modulate RNA polymerase II transcription.}, journal = {Nature Communications}, volume = {14}, year = {2023}, month = {2023 May 18}, pages = {2855}, abstract = {

NDP52 is an autophagy receptor involved in the recognition and degradation of invading pathogens and damaged organelles. Although NDP52 was first identified in the nucleus and is expressed throughout the cell, to date, there is no clear nuclear functions for NDP52. Here, we use a multidisciplinary approach to characterise the biochemical properties and nuclear roles of NDP52. We find that NDP52 clusters with RNA Polymerase II (RNAPII) at transcription initiation sites and that its overexpression promotes the formation of additional transcriptional clusters. We also show that depletion of NDP52 impacts overall gene expression levels in two model mammalian cells, and that transcription inhibition affects the spatial organisation and molecular dynamics of NDP52 in the nucleus. This directly links NDP52 to a role in RNAPII-dependent transcription. Furthermore, we also show that NDP52 binds specifically and with high affinity to double-stranded DNA (dsDNA) and that this interaction leads to changes in DNA structure in vitro. This, together with our proteomics data indicating enrichment for interactions with nucleosome remodelling proteins and DNA structure regulators, suggests a possible function for NDP52 in chromatin regulation. Overall, here we uncover nuclear roles for NDP52 in gene expression and DNA structure regulation.

}, keywords = {Animals, Autophagy, DNA, Mammals, Nuclear Proteins, Nucleic Acid Conformation, RNA Polymerase II}, issn = {2041-1723}, doi = {10.1038/s41467-023-38572-9}, author = {Dos Santos, {\'A}lia and Rollins, Daniel E and Hari-Gupta, Yukti and McArthur, Hannah and Du, Mingxue and Ru, Sabrina Yong Zi and Pidlisna, Kseniia and Stranger, Ane and Lorgat, Faeeza and Lambert, Danielle and Brown, Ian and Howland, Kevin and Aaron, Jesse and Wang, Lin and Ellis, Peter J I and Chew, Teng-Leong and Martin-Fernandez, Marisa and Pyne, Alice L B and Toseland, Christopher P} } @article {68959, title = {Dissecting Cell Plate Development During Plant Cytokinesis.}, journal = {Microscopy and Microanalysis}, volume = {29}, year = {2023}, month = {2023 Jul 22}, pages = {865}, keywords = {Cytokinesis}, issn = {1435-8115}, doi = {10.1093/micmic/ozad067.428}, author = {Sinclair, Rosalie and Cox, Daniel and Heddleston, John and Aaron, Jesse and Wait, Eric and Wilkop, Thomas and Drakakaki, Georgia} } @article {68934, title = {Investigating the composition and recruitment of the mycobacterial ImuA{\textquoteright}-ImuB-DnaE2 mutasome.}, journal = {eLife}, volume = {12}, year = {2023}, month = {2023 Aug 02}, abstract = {

A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded {\textquoteright}mycobacterial mutasome{\textquoteright} - minimally comprising DnaE2 polymerase and ImuA{\textquoteright} and ImuB accessory proteins - remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III β subunit (β clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an mutant containing a disrupted β clamp-binding motif abolishes ImuB-β clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this β clamp-binding antibiotic collapses pre-formed ImuB-β clamp complexes. These observations establish the essentiality of the ImuB-β clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.

}, keywords = {Antitubercular Agents, Bacterial Proteins, DNA Repair, Mutagenesis, Mycobacterium tuberculosis}, issn = {2050-084X}, doi = {10.7554/eLife.75628}, author = {Gessner, Sophia and Martin, Zela Alexandria-Mae and Reiche, Michael A and Santos, Joana A and Dinkele, Ryan and Ramudzuli, Atondaho and Dhar, Neeraj and de Wet, Timothy J and Anoosheh, Saber and Lang, Dirk M and Aaron, Jesse and Chew, Teng-Leong and Herrmann, Jennifer and M{\"u}ller, Rolf and McKinney, John D and Woodgate, Roger and Mizrahi, Valerie and Venclovas, {\v C}eslovas and Lamers, Meindert H and Warner, Digby F} } @article {67963, title = {Myosin VI regulates the spatial organisation of mammalian transcription initiation.}, journal = {Nature Communications}, volume = {13}, year = {2022}, month = {2022 Mar 15}, pages = {1346}, abstract = {

During transcription, RNA Polymerase II (RNAPII) is spatially organised within the nucleus into clusters that correlate with transcription activity. While this is a hallmark of genome regulation in mammalian cells, the mechanisms concerning the assembly, organisation and stability remain unknown. Here, we have used combination of single molecule imaging and genomic approaches to explore the role of nuclear myosin VI (MVI) in the nanoscale organisation of RNAPII. We reveal that MVI in the nucleus acts as the molecular anchor that holds RNAPII in high density clusters. Perturbation of MVI leads to the disruption of RNAPII localisation, chromatin organisation and subsequently a decrease in gene expression. Overall, we uncover the fundamental role of MVI in the spatial regulation of gene expression.

}, issn = {2041-1723}, doi = {10.1038/s41467-022-28962-w}, author = {Hari-Gupta, Yukti and Fili, Natalia and Dos Santos, {\'A}lia and Cook, Alexander W and Gough, Rosemarie E and Reed, Hannah C W and Wang, Lin and Aaron, Jesse and Venit, Tomas and Wait, Eric and Grosse-Berkenbusch, Andreas and Gebhardt, J Christof M and Percipalle, Piergiorgio and Chew, Teng-Leong and Martin-Fernandez, Marisa and Toseland, Christopher P} } @article {68412, title = {Single molecule microscopy to profile the effect of zinc status on transcription factor dynamics.}, journal = {Scientific Reports}, volume = {12}, year = {2022}, month = {2022 Oct 22}, pages = {17789}, abstract = {

The regulation of transcription is a complex process that involves binding of transcription factors (TFs) to specific sequences, recruitment of cofactors and chromatin remodelers, assembly of the pre-initiation complex and 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 tracking individual TF molecules as they diffuse in the nucleus and interact with DNA. Here we employ multifocus microscopy and highly inclined 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. We used 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 GR was largely insensitive to perturbations of zinc, whereas CTCF was significantly affected by zinc depletion and its dwell time was affected by zinc elevation. These results indicate that at least some transcription factors are sensitive to zinc dynamics, revealing a potential new layer of transcriptional regulation.

}, keywords = {CCCTC-Binding Factor, Chromatin, DNA, Receptors, Glucocorticoid, RNA Polymerase II, Single Molecule Imaging, Transcription Factors, Zinc}, issn = {2045-2322}, doi = {10.1038/s41598-022-22634-x}, author = {Damon, Leah J and Aaron, Jesse and Palmer, Amy E} } @article {68101, title = {Super-resolution visualization of chromatin loop folding in human lymphoblastoid cells using interferometric photoactivated localization microscopy.}, journal = {Scientific Reports}, volume = {12}, year = {2022}, month = {2022 May 20}, pages = {8582}, abstract = {

The three-dimensional (3D) genome structure plays a fundamental role in gene regulation and cellular functions. Recent studies in 3D genomics inferred the very basic functional chromatin folding structures known as chromatin loops, the long-range chromatin interactions that are mediated by protein factors and dynamically extruded by cohesin. We combined the use of FISH staining of a very short (33\ kb) chromatin fragment, interferometric photoactivated localization microscopy (iPALM), and traveling salesman problem-based heuristic loop reconstruction algorithm from an image of the one of the strongest CTCF-mediated chromatin loops in human lymphoblastoid cells. In total, we have generated thirteen good quality images of the target chromatin region with 2-22\ nm oligo probe localization precision. We visualized the shape of the single chromatin loops with unprecedented genomic resolution which allowed us to study the structural heterogeneity of chromatin looping. We were able to compare the physical distance maps from all reconstructed image-driven computational models with contact frequencies observed by ChIA-PET and Hi-C genomic-driven methods to examine the concordance between single cell imaging and population based genomic data.

}, keywords = {Chromatin, Chromatin Immunoprecipitation Sequencing, Chromosomes, Genome, Humans, Microscopy}, issn = {2045-2322}, doi = {10.1038/s41598-022-12568-9}, author = {Parteka-Tojek, Zofia and Zhu, Jacqueline Jufen and Lee, Byoungkoo and Jodkowska, Karolina and Wang, Ping and Aaron, Jesse and Chew, Teng-Leong and Banecki, Krzysztof and Plewczy{\'n}ski, Dariusz and Ruan, Yijun} } @article {67711, title = {Campylobacter jejuni Triggers Signaling through Host Cell Focal Adhesions To Inhibit Cell Motility.}, journal = {mBio}, volume = {12}, year = {2021}, month = {2021 Aug 24}, pages = {e0149421}, abstract = {

Campylobacter jejuni is a major foodborne pathogen that exploits the focal adhesions of intestinal cells to promote invasion and cause severe gastritis. Focal adhesions are multiprotein complexes involved in bidirectional signaling between the actin cytoskeleton and the extracellular matrix. We investigated the dynamics of focal adhesion structure and function in C. jejuni-infected cells using a comprehensive set of approaches, including confocal microscopy of live and fixed cells, immunoblotting, and superresolution interferometric photoactivated localization microscopy (iPALM). We found that C. jejuni infection of epithelial cells results in increased focal adhesion size and altered topology. These changes resulted in a persistent modulatory effect on the host cell focal adhesion, evidenced by an increase in cell adhesion strength, a decrease in individual cell motility, and a reduction in collective cell migration. We discovered that C. jejuni infection causes an increase in phosphorylation of paxillin and an alteration of paxillin turnover at the focal adhesion, which together represent a potential mechanistic basis for altered cell motility. Finally, we observed that infection of epithelial cells with the C. jejuni wild-type strain in the presence of a protein synthesis inhibitor, a C. jejuni CadF and FlpA fibronectin-binding protein mutant, or a C. jejuni flagellar export mutant blunts paxillin phosphorylation and partially reestablishes individual host cell motility and collective cell migration. These findings provide a potential mechanism for the restricted intestinal repair observed in C. jejuni-infected animals and raise the possibility that bacteria targeting extracellular matrix components can alter cell behavior after binding and internalization by manipulating focal adhesions. Campylobacter jejuni is a major foodborne pathogen that causes severe gastritis. We investigated the dynamics of focal adhesion structure and function in C. jejuni-infected epithelial cells. Focal adhesions act as signaling complexes that connect the extracellular matrix to the intracellular cytoskeleton. The key findings of this study show that C. jejuni changes the structure (size and position), composition, and function of cellular focal adhesions using a combination of virulence factors. Mechanistically, we found that the changes in focal adhesion dynamics are dependent upon the activation of host cell signaling pathways, which affect the assembly and disassembly of cellular proteins from the focal adhesion. To summarize, we have identified a new cellular phenotype in C. jejuni-infected cells that may be responsible for the restricted intestinal repair observed in C. jejuni-infected animals.

}, issn = {2150-7511}, doi = {10.1128/mBio.01494-21}, author = {Klappenbach, Courtney M and Negretti, Nicholas M and Aaron, Jesse and Chew, Teng-Leong and Konkel, Michael E} } @article {67696, title = {Campylobacter jejuni Triggers Signaling through Host Cell Focal Adhesions To Inhibit Cell Motility.}, journal = {mBio}, year = {2021}, month = {2021 Aug 24}, pages = {e0149421}, abstract = {

Campylobacter jejuni is a major foodborne pathogen that exploits the focal adhesions of intestinal cells to promote invasion and cause severe gastritis. Focal adhesions are multiprotein complexes involved in bidirectional signaling between the actin cytoskeleton and the extracellular matrix. We investigated the dynamics of focal adhesion structure and function in C. jejuni-infected cells using a comprehensive set of approaches, including confocal microscopy of live and fixed cells, immunoblotting, and superresolution interferometric photoactivated localization microscopy (iPALM). We found that C. jejuni infection of epithelial cells results in increased focal adhesion size and altered topology. These changes resulted in a persistent modulatory effect on the host cell focal adhesion, evidenced by an increase in cell adhesion strength, a decrease in individual cell motility, and a reduction in collective cell migration. We discovered that C. jejuni infection causes an increase in phosphorylation of paxillin and an alteration of paxillin turnover at the focal adhesion, which together represent a potential mechanistic basis for altered cell motility. Finally, we observed that infection of epithelial cells with the C. jejuni wild-type strain in the presence of a protein synthesis inhibitor, a C. jejuni CadF and FlpA fibronectin-binding protein mutant, or a C. jejuni flagellar export mutant blunts paxillin phosphorylation and partially reestablishes individual host cell motility and collective cell migration. These findings provide a potential mechanism for the restricted intestinal repair observed in C. jejuni-infected animals and raise the possibility that bacteria targeting extracellular matrix components can alter cell behavior after binding and internalization by manipulating focal adhesions. Campylobacter jejuni is a major foodborne pathogen that causes severe gastritis. We investigated the dynamics of focal adhesion structure and function in C. jejuni-infected epithelial cells. Focal adhesions act as signaling complexes that connect the extracellular matrix to the intracellular cytoskeleton. The key findings of this study show that C. jejuni changes the structure (size and position), composition, and function of cellular focal adhesions using a combination of virulence factors. Mechanistically, we found that the changes in focal adhesion dynamics are dependent upon the activation of host cell signaling pathways, which affect the assembly and disassembly of cellular proteins from the focal adhesion. To summarize, we have identified a new cellular phenotype in C. jejuni-infected cells that may be responsible for the restricted intestinal repair observed in C. jejuni-infected animals.

}, issn = {2150-7511}, doi = {10.1128/mBio.01494-21}, author = {Klappenbach, Courtney M and Negretti, Nicholas M and Aaron, Jesse and Chew, Teng-Leong and Konkel, Michael E} } @article {67233, title = {ER-to-Golgi protein delivery through an interwoven, tubular network extending from ER.}, journal = {Cell}, volume = {184}, year = {2021}, month = {2021 Apr 29}, pages = {2412}, abstract = {

Cellular versatility depends on accurate trafficking of diverse proteins to their organellar destinations. For the secretory pathway (followed by approximately 30\% of all proteins), the physical nature of the vessel conducting the first portage (endoplasmic reticulum [ER] to Golgi apparatus) is unclear. We provide a dynamic 3D view of early secretory compartments in mammalian cells with isotropic resolution and precise protein localization using whole-cell, focused ion beam scanning electron microscopy with cryo-structured illumination microscopy and live-cell synchronized cargo release approaches. Rather than vesicles alone, the ER spawns an elaborate, interwoven tubular network of contiguous lipid bilayers (ER exit site) for protein export. This receptacle is capable of extending microns along microtubules while still connected to the ER by a thin neck. COPII localizes to this neck region and dynamically regulates cargo entry from the ER, while COPI acts more distally, escorting the detached, accelerating tubular entity on its way to joining the Golgi apparatus through microtubule-directed movement.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2021.03.035}, author = {Weigel, Aubrey V and Chang, Chi-Lun and Shtengel, Gleb and Xu, C Shan and Hoffman, David P and Freeman, Melanie and Iyer, Nirmala and Aaron, Jesse and Khuon, Satya and Bogovic, John and Qiu, Wei and Hess, Harald F and Lippincott-Schwartz, Jennifer} } @article {67200, title = {A guide to accurate reporting in digital image processing - can anyone reproduce your quantitative analysis?}, journal = {Journal of Cell Science}, volume = {134}, year = {2021}, month = {2021 Mar 30}, abstract = {

Considerable attention has been recently paid to improving replicability and reproducibility in life science research. This has resulted in commendable efforts to standardize a variety of reagents, assays, cell lines and other resources. However, given that microscopy is a dominant tool for biologists, comparatively little discussion has been offered regarding how the proper reporting and documentation of microscopy relevant details should be handled. Image processing is a critical step of almost any microscopy-based experiment; however, improper, or incomplete reporting of its use in the literature is pervasive. The chosen details of an image processing workflow can dramatically determine the outcome of subsequent analyses, and indeed, the overall conclusions of a study. This Review aims to illustrate how proper reporting of image processing methodology improves scientific reproducibility and strengthens the biological conclusions derived from the results.

}, issn = {1477-9137}, doi = {10.1242/jcs.254151}, author = {Aaron, Jesse and Chew, Teng-Leong} } @article {67670, title = {Imaging Africa: a strategic approach to optical microscopy training in Africa.}, journal = {Nature Methods}, volume = {18}, year = {2021}, month = {2021 Aug 20}, pages = {847-855}, issn = {1548-7105}, doi = {10.1038/s41592-021-01227-y}, author = {Reiche, Michael A and Warner, Digby F and Aaron, Jesse and Khuon, Satya and Fletcher, Daniel A and Hahn, Klaus and Rogers, Kelly L and Mhlanga, Musa and Koch, Anastasia and Quaye, Wendye and Chew, Teng-Leong} } @article {67709, title = {Regulated exocytosis: Renal Aquaporin-2 3D Vesicular Network Organization and Association with F-actin.}, journal = {American Journal of Physiology: Cell Physiology}, volume = {321}, year = {2021}, month = {2021 Dec 01}, abstract = {

Regulated vesicle exocytosis is a key response to extracellular stimuli in diverse physiological processes; including hormone regulated short-term urine concentration. In the renal collecting duct, the water channel aquaporin-2 localizes to the apical plasma membrane as well as small, sub-apical vesicles. In response to stimulation with the antidiuretic hormone, arginine vasopressin, aquaporin-2 containing vesicles fuse with the plasma membrane, which increases collecting duct water reabsorption and thus, urine concentration. The nano-scale size of these vesicles has limited analysis of their 3D organization. Using a cell system combined with 3D super resolution microscopy, we provide the first direct analysis of the 3D network of aquaporin-2 containing exocytic vesicles in a cell culture system. We show that aquaporin-2 vesicles are 43 {\textpm} 3nm in diameter, a size similar to synaptic vesicles, and that one fraction of AQP2 vesicles localized with the sub-cortical F-actin layer and the other localized in between the F-actin layer and the plasma membrane. Aquaporin-2 vesicles associated with F-actin and this association was enhanced in a serine 256 phospho-mimic of aquaporin-2, whose phosphorylation is a key event in antidiuretic hormone-mediated aquaporin-2 vesicle exocytosis.

}, issn = {1522-1563}, doi = {10.1152/ajpcell.00255.2021}, author = {Holst, Mikkel R and Gammelgaard, Louis and Aaron, Jesse and Login, Fr{\'e}d{\'e}ric H and Rajkumar, Sampavi and Hahn, Ute and Nejsum, Lene N} } @article {65904, title = {DNA damage alters nuclear mechanics through chromatin reorganization.}, journal = {Nucleic Acids Research}, year = {2020}, month = {2020 Dec 16}, abstract = {

DNA double-strand breaks drive genomic instability. However, it remains unknown how these processes may affect the biomechanical properties of the nucleus and what role nuclear mechanics play in DNA damage and repair efficiency. Here, we have used Atomic Force Microscopy to investigate nuclear mechanical changes, arising from externally induced DNA damage. We found that nuclear stiffness is significantly reduced after cisplatin treatment, as a consequence of DNA damage signalling. This softening was linked to global chromatin decondensation, which improves molecular diffusion within the organelle. We propose that this can increase recruitment for repair factors. Interestingly, we also found that reduction of nuclear tension, through cytoskeletal relaxation, has a protective role to the cell and reduces accumulation of DNA damage. Overall, these changes protect against further genomic instability and promote DNA repair. We propose that these processes may underpin the development of drug resistance.

}, issn = {1362-4962}, doi = {10.1093/nar/gkaa1202}, author = {Dos Santos, {\'A}lia and Cook, Alexander W and Gough, Rosemarie E and Schilling, Martin and Olszok, Nora A and Brown, Ian and Wang, Lin and Aaron, Jesse and Martin-Fernandez, Marisa L and Rehfeldt, Florian and Toseland, Christopher P} } @article {65694, title = {A post-invasion role for type III effector TarP in modulating the dynamics and organization of host cell focal adhesions.}, journal = {Journal of Biological Chemistry}, volume = {295}, year = {2020}, month = {2020 Oct 23}, pages = {14763-14779}, abstract = {

The human pathogen targets epithelial cells lining the genital mucosa. We observed that infection of various cell types, including fibroblasts and epithelial cells resulted in the formation of unusually stable and mature focal adhesions that resisted disassembly induced by the myosin II inhibitor, blebbistatin. Super-resolution microscopy revealed in infected cells the vertical displacement of paxillin and FAK from the signaling layer of focal adhesions; while vinculin remained in its normal position within the force transduction layer. The candidate type III effector TarP which localized to focal adhesions during infection and when expressed ectopically, was sufficient to mimic both the reorganization and blebbistatin-resistant phenotypes. These effects of TarP, including its localization to focal adhesions, required a post-invasion interaction with the host protein vinculin through a specific domain at the C-terminus of TarP. This interaction is repurposed from an actin-recruiting and -remodeling complex to one that mediates nano-architectural and dynamic changes of focal adhesions. The consequence of -stabilized focal adhesions was restricted cell motility and enhanced attachment to the extracellular matrix. Thus, via a novel mechanism, inserts TarP within focal adhesions to alter their organization and stability.

}, issn = {1083-351X}, doi = {10.1074/jbc.RA120.015219}, author = {Pedrosa, Antonio T and Murphy, Korinn N and Nogueira, Ana T and Brinkworth, Amanda J and Thwaites, Tristan R and Aaron, Jesse and Chew, Teng-Leong and Carabeo, Rey A} } @article {65616, title = {Super-Resolution Fluorescence Imaging Reveals That Serine Incorporator Protein 5 Inhibits Human Immunodeficiency Virus Fusion by Disrupting Envelope Glycoprotein Clusters.}, journal = {ACS Nano}, year = {2020}, month = {2020 May 22}, abstract = {

Serine incorporator protein 5 (SERINC5) is the host anti-retroviral factor that reduces HIV-1 infectivity by incorporating into virions and inhibiting the envelope glycoprotein (Env) mediated virus fusion with target cells. We and others have shown that SERINC5 incorporation into virions alters the Env structure and sensitizes the virus to broadly neutralizing antibodies targeting cryptic Env epitopes. We have also found that SERINC5 accelerates the loss of Env function over time compared to control viruses. However, the exact mechanism by which SERINC5 inhibits HIV-1 fusion is not understood. Here, we utilized 2D and 3D super-resolution microscopy to examine the effect of SERINC5 on the distribution of Env glycoproteins on single HIV-1 particles. We find that, in agreement with a previous report, Env glycoproteins form clusters on the surface of mature virions. Importantly, incorporation of SERINC5, but not SERINC2, which lacks antiviral activity, disrupted Env clusters without affecting the overall Env content. We also show that SERINC5 and SERINC2 also form clusters on single virions. Unexpectedly, Env and SERINCs molecules exhibited poor co-distribution on virions, as evidenced by much greater Env-SERINC pairwise distances compare to Env-Env distances. This observation is inconsistent with the previously reported interaction between Env and SERINC5 and suggests an indirect effect of SERINC5 on Env cluster formation. Collectively, our results reveal a multifaceted mechanism of SERINC5-mediated restriction of HIV-1 fusion that, aside from the effects on individual Env trimers, involves disruption of Env clusters, which likely serve as sites of viral fusion with target cells.

}, issn = {1936-086X}, doi = {10.1021/acsnano.0c02699}, author = {Chen, Yen-Cheng and Sood, Chetan and Marin, Mariana and Aaron, Jesse and Gratton, Enrico and Salaita, Khalid and Melikyan, Gregory B} } @article {65603, title = {Ultrastructural visualization of 3D chromatin folding using volume electron microscopy and DNA in situ hybridization.}, journal = {Nature Communications}, volume = {11}, year = {2020}, month = {2020 May 01}, pages = {2120}, abstract = {

The human genome is extensively folded into 3-dimensional organization. However, the detailed 3D chromatin folding structures have not been fully visualized due to the lack of robust and ultra-resolution imaging capability. Here, we report the development of an electron microscopy method that combines serial block-face scanning electron microscopy with in situ hybridization (3D-EMISH) to visualize 3D chromatin folding at targeted genomic regions with ultra-resolution (5 {\texttimes} 5 {\texttimes} 30 nm in xyz dimensions) that is superior to the current super-resolution by fluorescence light microscopy. We apply 3D-EMISH to human lymphoblastoid cells at a 1.7 Mb segment of the genome and visualize a large number of distinctive 3D chromatin folding structures in ultra-resolution. We further quantitatively characterize the reconstituted chromatin folding structures by identifying sub-domains, and uncover a high level heterogeneity of chromatin folding ultrastructures in individual nuclei, suggestive of extensive dynamic fluidity in 3D chromatin states.

}, issn = {2041-1723}, doi = {10.1038/s41467-020-15987-2}, author = {Trzaskoma, Pawe{\l} and Ruszczycki, B{\l}a{\.z}ej and Lee, Byoungkoo and Pels, Katarzyna K and Krawczyk, Katarzyna and Bokota, Grzegorz and Szczepankiewicz, Andrzej A and Aaron, Jesse and Walczak, Agnieszka and {\'S}liwi{\'n}ska, Ma{\l}gorzata A and Magalska, Adriana and Kadlof, Michal and Wolny, Artur and Parteka, Zofia and Arabasz, Sebastian and Kiss-Arabasz, Magdalena and Plewczy{\'n}ski, Dariusz and Ruan, Yijun and Wilczy{\'n}ski, Grzegorz M} } @article {65127, title = {Differential nanoscale organisation of LFA-1 modulates T cell migration.}, journal = {Journal of Cell Science}, volume = {132}, year = {2019}, month = {2019 Aug 29}, pages = {1-28}, issn = {0021-9533}, doi = {10.1242/jcs.232991}, url = {http://jcs.biologists.org/lookup/doi/10.1242/jcs.23299}, author = {Shannon, Michael J. and Pineau, Judith and Griffi{\'e}, Juliette and Aaron, Jesse and Peel, Tamlyn and Williamson, David J. and Zamoyska, Rose and Cope, Andrew P. and Cornish, Georgina H. and Owen, Dylan M.} } @article {64903, title = {Practical considerations in particle and object tracking and analysis.}, journal = {Current Protocols in Cell Biology}, volume = {83}, year = {2019}, month = {2019 Jun 01}, pages = {e88}, abstract = {

The rapid advancement of live-cell imaging technologies has enabled biologists to generate high-dimensional data to follow biological movement at the microscopic level. Yet, the "perceived" ease of use of modern microscopes has led to challenges whereby sub-optimal data are commonly generated that cannot support quantitative tracking and analysis as a result of various ill-advised decisions made during image acquisition. Even optimally acquired images often require further optimization through digital processing before they can be analyzed. In writing this article, we presume our target audience to be biologists with a foundational understanding of digital image acquisition and processing, who are seeking to understand the essential steps for particle/object tracking experiments. It is with this targeted readership in mind that we review the basic principles of image-processing techniques as well as analysis strategies commonly used for tracking experiments. We conclude this technical survey with a discussion of how movement behavior can be mathematically modeled and described. {\textcopyright} 2019 by John Wiley \& Sons, Inc.

}, issn = {1934-2616}, doi = {10.1002/cpcb.88}, author = {Aaron, Jesse and Wait, Eric and DeSantis, Michael and Chew, Teng-Leong} } @article {64544, title = {Rhomboid distorts lipids to break the viscosity-imposed speed limit of membrane diffusion.}, journal = {Science (New York, N.Y.)}, volume = {363}, year = {2019}, month = {2019 Feb 01}, abstract = {

Enzymes that cut proteins inside membranes regulate diverse cellular events, including cell signaling, homeostasis, and host-pathogen interactions. Adaptations that enable catalysis in this exceptional environment are poorly understood. We visualized single molecules of multiple rhomboid intramembrane proteases and unrelated proteins in living cells (human and ) and planar lipid bilayers. Notably, only rhomboid proteins were able to diffuse above the Saffman-Delbr{\"u}ck viscosity limit of the membrane. Hydrophobic mismatch with the irregularly shaped rhomboid fold distorted surrounding lipids and propelled rhomboid diffusion. The rate of substrate processing in living cells scaled with rhomboid diffusivity. Thus, intramembrane proteolysis is naturally diffusion-limited, but cells mitigate this constraint by using the rhomboid fold to overcome the "speed limit" of membrane diffusion.

}, issn = {1095-9203}, doi = {10.1126/science.aao0076}, author = {Kreutzberger, Alex J B and Ji, Ming and Aaron, Jesse and Mihaljevi{\'c}, Ljubica and Urban, Sini{\v s}a} } @article {65197, title = {Superresolution architecture of cornerstone focal adhesions in human pluripotent stem cells.}, journal = {Nature Communications}, volume = {10}, year = {2019}, month = {2019 Oct 18}, pages = {4756}, abstract = {

While it is clear that key transcriptional programmes are important for maintaining pluripotency, the requirement for cell adhesion to the extracellular matrix remains poorly defined. Human pluripotent stem cells\ (hPSCs) form colonies encircled by an actin ring and large stable cornerstone focal adhesions (FA). Using superresolution two-colour interferometric photo-activated localisation microscopy, we examine the three-dimensional architecture of cornerstone adhesions and report vertical lamination of FA proteins with three main structural features distinct from previously studied focal adhesions: 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 previously reported, displaying a head-above-tail orientation, and 3) surprisingly, actin and α-actinin are present in two discrete z-layers. Finally, we report that depletion of kanks diminishes FA patterning, and actin organisation within the colony, indicating a role for kanks in hPSC colony architecture.

}, issn = {2041-1723}, doi = {10.1038/s41467-019-12611-w}, author = {Stubb, Aki and Guzm{\'a}n, Camilo and N{\"a}rv{\"a}, Elisa and Aaron, Jesse and Chew, Teng-Leong and Saari, Markku and Miihkinen, Mitro and Jacquemet, Guillaume and Ivaska, Johanna} } @article {49341, title = {The actin cytoskeletal architecture of estrogen receptor positive breast cancer cells suppresses invasion.}, journal = {Nature Communications}, volume = {9}, year = {2018}, month = {2018 Jul 30}, pages = {2980}, abstract = {

Estrogen promotes growth of estrogen receptor-positive (ER+) breast tumors. However, epidemiological studies examining the prognostic characteristics of breast cancer in postmenopausal women receiving hormone replacement therapy reveal a significant decrease in tumor dissemination, suggesting that estrogen has potential protective effects against cancer cell invasion. Here, we show that estrogen suppresses invasion of ER+ breast cancer cells by increasing transcription of the Ena/VASP protein, EVL, which promotes the generation of suppressive cortical actin bundles that inhibit motility dynamics, and is crucial for the ER-mediated suppression of invasion in vitro and in vivo. Interestingly, despite its benefits in suppressing tumor growth, anti-estrogenic endocrine therapy decreases EVL expression and increases local invasion in patients. Our results highlight the dichotomous effects of estrogen on tumor progression and suggest that, in contrast to its established role in promoting growth of ER+ tumors, estrogen has a significant role in suppressing invasion through actin cytoskeletal remodeling.

}, issn = {2041-1723}, doi = {10.1038/s41467-018-05367-2}, author = {Padilla-Rodriguez, Marco and Parker, Sara S and Adams, Deanna G and Westerling, Thomas and Puleo, Julieann I and Watson, Adam W and Hill, Samantha M and Noon, Muhammad and Gaudin, Rapha{\"e}l and Aaron, Jesse and Tong, Daoqin and Roe, Denise J and Knudsen, Beatrice and Mouneimne, Ghassan} } @article {49021, title = {The Chlamydia type III effector TarP alters the dynamics and organization of host focal adhesions.}, journal = {bioRxiv}, year = {2018}, month = {2018 Jan 23}, abstract = {

Bacterial infection of mucosal epithelial cells triggers cell exfoliation to limit the dissemination of infection within the tissue. Therefore, mucosal pathogens must possess strategies to counteract cell extrusion in response to infection. Chlamydia trachomatis spends most of its intracellular development in the non-infectious form. Thus, premature host cell extrusion is detrimental to the pathogen. We demonstrate that C. trachomatis alters the dynamics of focal adhesions. Live-cell microscopy showed that focal adhesions in C. trachomatis-infected cells displayed increased stability. In contrast, focal adhesions in mock-infected cells readily disassembled upon inhibition of myosin II by blebbisttin. Super-resolution microscopy revealed a reorganization of paxillin and FAK in infected cells. Ectopically expressed type III effector TarP localized to focal adhesions, leading to their stabilization and reorganization in a vinculin-dependent manner. Overall, the results indicate that C. trachomatis possesses a dedicated mechanism to regulate host cell focal adhesion dynamics.

}, doi = {10.1101/250563}, author = {Pedrosa, Antonio T and Nogueira, Ana T and Thwaites, Tristan R and Aaron, Jesse and Chew, Teng-Leong and Carabeo, Rey A} } @article {49467, title = {Development of 2-colour and 3D SMLM data analysis methods for fibrous spatial point patterns.}, journal = {Journal of Physics D: Applied Physics}, volume = {52}, year = {2018}, month = {2018 Oct 11}, pages = {1}, abstract = {

Abstract ingle molecule localisation microscopy (SMLM), experimentally achieved over a decade ago, has become a routinely used analytical tool across the life sciences. Synergistic advances in probe chemistry, optical physics and data analysis has propelled SMLM into the quantitative realm, enabling unprecedented access to the cellular machinery at the nanoscale. In its early years, SMLM primarily served as a platform for impressive rendered images of sub diffraction scale structures, however more recently a shift towards interrogating SMLM point pattern data in a robust mathematical framework has occurred. A prevalent theme in the SMLM field is the need for quantitative analytical methods, to better understand the underlying processes on which SMLM reports and to extract statistically valid biological insights. Whilst some forms of post processing analytics, for example cluster analysis, have been widely studied, others such as fibre analysis remain in their infancy. Here, we review the current state of the art of cluster analysis and fibre analysis and present new methods for their implementation in both 3D SMLM data sets and multi-colour data.

}, doi = {10.1088/1361-6463/aae7ac}, author = {Peters, Ruby and Griffi{\'e}, Juliette and Williamson, David and Aaron, Jesse and Khuon, Satya and Owen, Dylan} } @article {49051, title = {Measuring integrin conformational change on the cell surface with super-resolution microscopy.}, journal = {Cell Reports}, volume = {22}, year = {2018}, month = {2018 Feb 13}, pages = {1903-1912}, abstract = {

We use super-resolution interferometric photoactivation and localization microscopy (iPALM) and a constrained photoactivatable fluorescent protein integrin fusion to measure the displacement of the head of integrin lymphocyte function-associated 1 (LFA-1) resulting from integrin conformational change on the cell surface. We demonstrate that the distance of the LFA-1 head increases substantially between basal and ligand-engaged conformations, which can only be explained at the molecular level by integrin extension. We further demonstrate that one class of integrin antagonist maintains the bent conformation, while another antagonist class induces extension. Our molecular scale measurements on cell-surface LFA-1 are in excellent agreement with distances derived from crystallographic and electron microscopy structures of bent and extended integrins. Our distance measurements are also in excellent agreement with a previous model of LFA-1 bound to ICAM-1 derived from the orientation of LFA-1 on the cell surface measured using fluorescence polarization microscopy.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2018.01.062}, author = {Moore, Travis I and Aaron, Jesse and Chew, Teng-Leong and Springer, Timothy A} } @article {49085, title = {Model-free quantification and visualization of colocalization in fluorescence images.}, journal = {Cytometry. Part A : the journal of the International Society for Analytical Cytology}, year = {2018}, month = {2018 Mar 13}, abstract = {

The spatial association between fluorescently tagged biomolecules in situ provides valuable insight into their biological relationship. Within the limits of diffraction, such association can be measured using either Pearson{\textquoteright}s Correlation Coefficient (PCC) or Spearman{\textquoteright}s Rank Coefficient (SRC), which are designed to measure linear and monotonic correlations, respectively. However, the relationship between real biological signals is often more complex than these measures assume, rendering their results difficult to interpret. Here, we have adapted methods from the field of information theory to measure the association between two probes{\textquoteright} concentrations based on their statistical dependence. Our approach is mathematically more general than PCC or SRC, making no assumptions about the type of relationship between the probes. We show that when applied to biological images, our measures provide more intuitive results that are also more robust to outliers and the presence of multiple relationships than PCC or SRC. We also devise a display technique to highlight regions in the input images where the probes{\textquoteright} association is higher versus lower. We expect that our methods will allow biologists to more accurately and robustly quantify and visualize the association between two probes in a pair of fluorescence images. {\textcopyright} 2018 International Society for Advancement of Cytometry.

}, issn = {1552-4930}, doi = {10.1002/cyto.a.23356}, author = {Taylor, Aaron B and Ioannou, Maria S and Aaron, Jesse and Chew, Teng-Leong} } @article {49221, title = {Single molecule fate of HIV-1 envelope reveals late-stage viral lattice incorporation.}, journal = {Nature Communications}, volume = {9}, year = {2018}, month = {2018 May 10}, pages = {1861}, abstract = {

Human immunodeficiency virus type 1 (HIV-1) assembly occurs on the inner leaflet of the host cell plasma membrane, incorporating the essential viral envelope glycoprotein (Env) within a budding lattice of HIV-1 Gag structural proteins. The mechanism by which Env incorporates into viral particles remains poorly understood. To determine the mechanism of recruitment of Env to assembly sites, we interrogate the subviral angular distribution of Env on cell-associated virus using multicolor, three-dimensional (3D) superresolution microscopy. We demonstrate that, in a manner dependent on cell type and on the long cytoplasmic tail of Env, the distribution of Env is biased toward the necks of cell-associated particles. We postulate that this neck-biased distribution is regulated by vesicular retention and steric complementarity of Env during independent Gag lattice formation.

}, issn = {2041-1723}, doi = {10.1038/s41467-018-04220-w}, author = {Buttler, Carmen A and Pezeshkian, Nairi and Fernandez, Melissa V and Aaron, Jesse and Norman, Sofya and Freed, Eric O and Van Engelenburg, Schuyler B} } @article {48621, title = {3D Bayesian cluster analysis of super-resolution data reveals LAT recruitment to the T cell synapse.}, journal = {Scientific Reports}, volume = {7}, year = {2017}, month = {2017 Jun 22}, pages = {4077}, abstract = {

Single-molecule localisation microscopy (SMLM) allows the localisation of fluorophores with a precision of 10-30 nm, revealing the cell{\textquoteright}s nanoscale architecture at the molecular level. Recently, SMLM has been extended to 3D, providing a unique insight into cellular machinery. Although cluster analysis techniques have been developed for 2D SMLM data sets, few have been applied to 3D. This lack of quantification tools can be explained by the relative novelty of imaging techniques such as interferometric photo-activated localisation microscopy (iPALM). Also, existing methods that could be extended to 3D SMLM are usually subject to user defined analysis parameters, which remains a major drawback. Here, we present a new open source cluster analysis method for 3D SMLM data, free of user definable parameters, relying on a model-based Bayesian approach which takes full account of the individual localisation precisions in all three dimensions. The accuracy and reliability of the method is validated using simulated data sets. This tool is then deployed on novel experimental data as a proof of concept, illustrating the recruitment of LAT to the T-cell immunological synapse in data acquired by iPALM providing ~10 nm isotropic resolution.

}, issn = {2045-2322}, doi = {10.1038/s41598-017-04450-w}, author = {Griffi{\'e}, Juliette and Shlomovich, Leigh and Williamson, David J and Shannon, Michael and Aaron, Jesse and Khuon, Satya and L Burn, Garth and Boelen, Lies and Peters, Ruby and Cope, Andrew P and Cohen, Edward A K and Rubin-Delanchy, Patrick and Owen, Dylan M} }