Beatson Advanced Imaging Resource (BAIR)
Introduction
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Dr Leo CarlinHead of BAIR Facility |
Beatson Advanced Imaging Resource (BAIR) scientists, headed by Leo Carlin, work closely with the Institute's researchers to uncover and interrogate important molecular pathways in cancer. We train scientists in all stages of modern imaging research from advice on sample preparation, basic and advanced microscope operation and data acquisition through to quantitative image analysis and interpretation.
We have a wide range of specialised microscopes within the facility that provide users with the flexibility to approach their research using a diverse range of applications. For example: users have access to a variety of confocal and widefield microscopes, super-resolution microscopes, high-content screening, in vivo imaging systems, and equipment for advanced techniques such as TIRF, FLIM/FRET and Multiphoton imaging.
Leo Carlin also leads the Leukocyte Dynamics group.
Image Credits - Top Left: Frederic Fercoq, Top Right: Nikki R. Paul, Bottom Left: Sophie Claydon, Bottom Right: Ed Roberts
Group Members
Peter Thomasonp.thomason@crukscotlandinstitute.ac.uk Confocal Microscopy |
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Lynn McGarryl.mcgarry@crukscotlandinstitute.ac.uk High Content Screening |
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Nikki Pauln.paul@crukscotlandinstitute.ac.uk Advanced Application Development |
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Ryan Corbynr.corbyn@crukscotlandinstitute.ac.uk Image Analysis |
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Claire Mitchellc.mitchell@crukscotlandinstitute.ac.uk General Microscopy |
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Beatrice Botturab.bottura@crukscotlandinstitute.ac.uk General Microscopy |
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Equipment
Confocal Microscopes
Zeiss LSM 880 with Airyscan
Image credit: Nikki Paul
The Zeiss LSM 880 with Airyscan is a super-resolution-capable inverted confocal microscope. It has a full cage environmental chamber for live cell imaging and is also suitable for photoactivation, FRAP and FCS.
Zeiss LSM 710
The Zeiss LSM 710 is equipped with the Zeiss Quasar detector configured with 2, 3 or 34 channels. Built around a Zeiss Axioimager upright stand the system is primarily for fixed samples. It is suitable for a wide range of applications including imaging of quadrupled labelled specimens, colocalisation of proteins, and 3D imaging experiments.
Nikon A1R
Image credit: Amelie Juin
A fully automated, highly sensitive confocal microscope system, the Nikon A1R is built around a Nikon Ti Eclipse microscope with (Perfect Focus System) PFS. The system has a full cage environmental chamber and is therefore suitable for live cell timelapse imaging.
The Nikon A1R has two scanning systems: Firstly, there is a conventional sawtooth scanning system – galvano, which is used for high-resolution imaging where speed is not essential. Secondly, there is a fast resonant scanner, which is used for applications where the need for speed outweighs the need for high resolution. The two scanning systems can be operated in rapid succession or simultaneously. This system is therefore also suitable for photoactivation and FRAP experiments.
Widefield Microscopes
Olympus BX51
The BX51 microscope is a widefield system available for bright field and fluorescence observation methods. It is a non-motorised upright microscope system which is used primarily for fixed histological tissue sections and cells. It is also used for fixed fluorescently labelled samples.
Nanosight LM10
The Nanosight LM10 allows rapid and accurate analysis of the size distribution and concentration of all types of nanoparticles from 10nm to 2000nm in diameter, depending on the instrument configuration and sample type. The nanosight can be used to detect exosomes and microvesicles which are secreted from cells and tissues.
Leica Laser Microdissection
Laser microdissection uses a laser to isolate specific microscopic regions from tissue samples. The system is built around a Leica DM6000B upright microscope stand. These samples can be further analysed using additional techniques such as proteomics and metabolomics.
Timelapse Microscopes
Nikon Long-Term Timelapse
Image credits: Savvas Nikolaou
BAIR has three Nikon TE 2000 Live-Cell Timelapse microscope systems with temperature control, CO2 and the Perfect Focus System (PFS). These systems are frequently used to image cell migration or cell division using phase/brightfield over time, and can also be used to detect fluorescent dyes or proteins.
Incucyte S3
The Incucyte S3 Live-Cell Imaging systems are automated microscopes that fit within tissue culture incubators. This permits long-term analysis of 2D and 3D cultures in multiple culture plate formats. The Incucyte S3 systems can also be used to detect fluorescent proteins or dyes, which are often used to measure cell proliferation and apoptosis. In combination with the WoundMaker tool, the Incucyte S3 systems are often used to monitor Scratch Wound Cell Migration over time in a 96-well plate format. The Incucyte S3 has additional software modules such as the Spheroid analysis module, which permits measurement of 3D tumouroids and organoids over time.
Super-Resolution Microscopes
Zeiss LSM 880 with Airyscan
Image credits: Savvas Nikolaou
The Zeiss LSM 880 with Airyscan is a super-resolution-capable confocal microscope. The 'Airyscan' detector delivers increased resolution (1.7x) with high sensitivity at around 140nm laterally and 400nm axially. The 'Airyscan Fast' mode increases scanning speeds by a factor of 4. This allows users to image at a higher speed without sacrificing sensitivity or resolution. It has a full cage environmental chamber for live cell imaging and is also suitable for photoactivation, FRAP and FCS.
Airyscan Principle: https://www.youtube.com/watch?v=QjKrLTHGnc8
Airyscan Fast: https://www.youtube.com/watch?v=ln6VUmLzLLc
Zeiss Multiphoton 880
The Zeiss Multiphoton 880 with Airyscan is a super-resolution-capable confocal microscope. This system is in an upright configuration, and is suited to in vivo imaging of e.g. skull/brain, lung sections and abdominal organs with the use of vacuum windows.
Image credits: Amanda McFarlane & Marco De Donatis
Elyra 7
The Zeiss Elyra 7 with Lattice SIM is a laser-based widefield microscope that permits rapid acquisition, coupling super-resolution microscopy with high speed. This microscope is already proving useful in a number of projects, including live-cell imaging of microtubule dynamics, intracellular trafficking, mitochondrial localisation and 3D imaging of Drosophila. In addition to Structured Illumination, this microscope is capable of fast optical sectioning in Apotome mode, and Single Molecule Localisation Microscopy (SMLM) using STORM or 3D-PALM. The recent SIM2 upgrade allows even faster acquisition and higher resolution processing.
Multiphoton Microscopes
Zeiss Multiphoton 880
The Zeiss Multiphoton 880 with Airyscan is a super-resolution-capable confocal microscope. This system is in an upright configuration, and is suited to in vivo imaging of e.g. skull/brain, lung sections and abdominal organs with the use of vacuum windows.
Trimscope 2
The LaVision TRIM microscope is a high-resolution multiphoton system able to image small groups of cells down to the level of subcellular structure and generate three-dimensional reconstructions. It is capable of fast and sectioned FLIM, as well as detecting second-harmonic generation (SHG).
This system is capable of in vivo imaging using e.g. abdominal or mammary windows. In addition, this system has dual channel FLIM detectors so can do simultaneous FLIM measurements of CFP and GFP-based biosensors.
FLIM/FRET
Andor SD FLIM
The Andor Revolution XD is built around a Nikon Ti- inverted microscope. This system has a Yokogawa CSU-X spinning disc confocal unit with a disk speed that supports up to 2,000 frames per second. The Lambert Instruments FLIM Attachment for lifetime imaging microscopy is a dedicated system that allows rapid image acquisition and generation of lifetime images in the frequency domain. To achieve this the system uses a modulated light source and a modulated image intensifier as a detector.
Trimscope 2
The LaVision TRIM microscope is a high-resolution multiphoton system able to image small groups of cells down to the level of subcellular structure and generate three-dimensional reconstructions. It is capable of fast and sectioned FLIM, as well as detecting second-harmonic generation (SHG). This system is capable of in vivo imaging using e.g. abdominal or mammary windows. In addition, this system has dual channel FLIM detectors so can do simultaneous FLIM measurements of CFP and GFP-based biosensors.
High-Content Analysis
The Screening Facility couples genetic and chemical high throughput screening with multiparametric phenotypic image analysis to support translational cancer research. We employ functional genomics (siRNA, shRNA, CRISPR) tools in both pooled and arrayed approaches to support target identification and target validation in multiple types of cancer. Our state-of-the-art image analysis capabilities, including a machine learning module, support identification of a suite of cellular phenotypes in both 2D and 3D environments.
Our Perkin Elmer Opera High-Content Screening system is supported by a robotic plate-loading arm, and JANUS and Wellmate liquid handling systems.
Opera Phenix
The Opera Phenix is a high-throughput confocal microscope primarily used for high-content screening and phenotype analysis. The system supports automated plate loading and image acquisition. The Opera Phenix is particulary advanced in 3D imaging of cells, organoids, spheroids and tissues. Coupled to Harmony/Columbus analysis software, the Opera Phenix can generate large amounts of data suitable for bioinformatics and machine learning.
Image credits: Emily Kay & Jessica Perochon
Please contact Lynn McGarry (l.mcgarry@crukscotlandinstitute.ac.uk) for information on High Content Screening.
Tissue Culture Microscopes
BAIR supports a number of tissue culture microscopes throughout the Beatson Institute. All these microscopes can be used to visualise fluorescent proteins in cells, and are coupled to a PC and camera.
Flow Cytometry
Cell Analysers
BD Fortessa – 5 laser, 20 parameter instrument utilised for simple to complex analysis of cell populations. Excellent for analysing the range of Brilliant Violet dyes with 6 being detected from the 405 laser.
Thermo Fisher Attune Nxt – 4 laser, 16 parameter instrument. Due to it’s unique Acoustic Focussing system it can analyse large volume samples quickly without loss of resolution. This instrument also has an Autosampler attached to it which allows the analysis of cells on 96 and 384 well plates.
BD Facsverse – 3 laser , 10 parameter machine used for the analysis of cell populations.
Cell Sorters
BD FacsAria III – 4 laser, 18 parameter instrument used to sort up to 4 distinct live cell populations into collection tubes or plates to allow these cells to be used for further experiments.
BD Fusion Sorter – 3 laser, 13 parameter instrument which is housed in a Class II containment hood which allows for the safe sorting of 4 distinct live populations of Category II cells (human and virus transfected) into collection tubes or plates for further analysis.
Please contact Tom Gilbey (t.gilbey@beatson.gla.ac.uk) for information of the flow cytometry equipment and analysis software.
Image Analysis
The BAIR Centre for Analysis and Visualisation of Experimental data is a dedicated analysis suite which gives users access to high-performance computers and both licenced and open-source software. Software includes Fiji, Imaris, Icy, Harmony, Zen, NIS Elements, Incucyte, Cell Profiler, QuPath, Metamorph, Flowjo.
Recent Publications
2024
Xavier, V.; Martinelli, S.; Corbyn, R.; Pennie, R.; Rakovic, K.; Powley, I. R.; Officer-Jones, L.; Ruscica, V.; Galloway, A.; Carlin, L. M.; Cowling, V. H.; Quesne, J. L.; Martinou, J.-C.; MacVicar, T. Mitochondrial Double-Stranded RNA Homeostasis Depends on Cell-Cycle Progression. Life Science Alliance 2024, 7 (11). https://doi.org/10.26508/lsa.202402764.
Thomason, P. A.; Corbyn, R.; Lilla, S.; Sumpton, D.; Gilbey, T.; Insall, R. H. Biogenesis of Lysosome-Related Organelles Complex-2 Is an Evolutionarily Ancient Proto-Coatomer Complex. Current Biology 2024, 34 (15), 3564-3581.e6. https://doi.org/10.1016/j.cub.2024.06.081.
Burgess, S. G.; Paul, N. R.; Richards, M. W.; Ault, J. R.; Askenatzis, L.; Claydon, S. G.; Corbyn, R.; Machesky, L. M.; Bayliss, R. A Nanobody Inhibitor of Fascin-1 Actin-Bundling Activity and Filopodia Formation. Open Biology 2024, 14 (3), 230376. https://doi.org/10.1098/rsob.230376.
2023
Whyte, D.; Skalka, G.; Walsh, P.; Wilczynska, A.; Paul, N. R.; Mitchell, C.; Nixon, C.; Clarke, W.; Bushell, M.; Morton, J. P.; Murphy, D. J.; Muthalagu, N. NUAK1 Governs Centrosome Replication in Pancreatic Cancer via MYPT1/PP1β and GSK3β-Dependent Regulation of PLK4. Mol Oncol 2023, 17 (7), 1212–1227. https://doi.org/10.1002/1878-0261.13425.
Schmidt, T.; Dabrowska, A.; Waldron, J. A.; Hodge, K.; Koulouras, G.; Gabrielsen, M.; Munro, J.; Tack, D. C.; Harris, G.; McGhee, E.; Scott, D.; Carlin, L. M.; Huang, D.; Le Quesne, J.; Zanivan, S.; Wilczynska, A.; Bushell, M. eIF4A1-Dependent mRNAs Employ Purine-Rich 5’UTR Sequences to Activate Localised eIF4A1-Unwinding through eIF4A1-Multimerisation to Facilitate Translation. Nucleic Acids Res 2023, 51 (4), 1859–1879.
Santi, A.; Kay, E.; Neilson, L.; McGarry, L.; Lilla, S.; Mullin, M.; Paul, N.; Fercoq, F.; Koulouras, G.; Blanco, G. R.; Athineos, D.; Mason, S.; Hughes, M.; Kieffer, Y.; Nixon, C.; Blyth, K.; Mechta-Grigoriou, F.; Carlin, L.; Zanivan, S. Cancer-Associated Fibroblasts Produce Matrix-Bound Vesicles That Influence Endothelial Cell Function. 2023. https://doi.org/10.1101/2023.01.13.523951.
Sandilands, E.; Freckmann, E. C.; Cumming, E. M.; Román-Fernández, A.; McGarry, L.; Anand, J.; Galbraith, L.; Mason, S.; Patel, R.; Nixon, C.; Cartwright, J.; Leung, H. Y.; Blyth, K.; Bryant, D. M. The Small GTPase ARF3 Controls Invasion Modality and Metastasis by Regulating N-Cadherin Levels. J Cell Biol 2023, 222 (4), e202206115. https://doi.org/10.1083/jcb.202206115.
Román-Fernández, A.; Mansour, M. A.; Kugeratski, F. G.; Anand, J.; Sandilands, E.; Galbraith, L.; Rakovic, K.; Freckmann, E. C.; Cumming, E. M.; Park, J.; Nikolatou, K.; Lilla, S.; Shaw, R.; Strachan, D.; Mason, S.; Patel, R.; McGarry, L.; Katoch, A.; Campbell, K. J.; Nixon, C.; Miller, C. J.; Leung, H. Y.; Le Quesne, J.; Norman, J. C.; Zanivan, S.; Blyth, K.; Bryant, D. M. Spatial Regulation of the Glycocalyx Component Podocalyxin Is a Switch for Prometastatic Function. Sci Adv 2023, 9 (5), eabq1858. https://doi.org/10.1126/sciadv.abq1858.
Raffo-Iraolagoitia, X.; McFarlane, A.; Kruspig, B.; Fercoq, F.; Secklehner, J.; De Donatis, M.; Mackey, J.; Wiesheu, R.; Laing, S.; Hsieh, Y.-C.; Shaw, R.; Corbyn, R.; Nixon, C.; Miller, C.; Kirschner, K.; Bain, C.; Murphy, D.; Coffelt, S.; Carlin, L. Γδ T Cells Impair Airway Macrophage Differentiation in Lung Adenocarcinoma. 2023. https://doi.org/10.1101/2023.09.14.557344.
Papalazarou, V.; Newman, A. C.; Huerta-Uribe, A.; Legrave, N. M.; Falcone, M.; Zhang, T.; McGarry, L.; Athineos, D.; Shanks, E.; Blyth, K.; Vousden, K. H.; Maddocks, O. D. K. Phenotypic Profiling of Solute Carriers Characterizes Serine Transport in Cancer. Nat Metab 2023. https://doi.org/10.1038/s42255-023-00936-2.
Nutt, K.; Olesker, D.; McGhee, E.; Hungerford, G.; Leburn, C.; Taylor, J. High-Efficiency Digitally Scanned Light-Sheet Fluorescence Lifetime Microscopy (DSLM-FLIM). 2023. https://doi.org/10.1101/2023.06.02.543377.
Nikolatou, K.; Sandilands, E.; Román-Fernández, A.; Cumming, E. M.; Freckmann, E.; Lilla, S.; Buetow, L.; McGarry, L.; Neilson, M.; Shaw, R.; Strachan, D.; Miller, C.; Huang, D. T.; McNeish, I. A.; Norman, J. C.; Zanivan, S.; Bryant, D. M. PTEN Deficiency Exposes a Requirement for an ARF GTPase Module for Integrin-Dependent Invasion in Ovarian Cancer. EMBO J 2023, 42 (18), e113987. https://doi.org/10.15252/embj.2023113987.
Moore, M.; Pardo-Fernandez, L.; Mitchell, L.; Schmidt, T.; Waldron, J.; May, S.; Muller, M.; Smith, R.; Strathdee, D.; Bryson, S.; Hodge, K.; Lilla, S.; Wilczynska, A.; McGarry, L.; Gillen, S.; Peter-Durairaj, R.; Kanellos, G.; Nixon, C.; Zanivan, S.; Sansom, O.; Bird, T.; Bushell, M.; Norman, J. The eIF4A2 Negative Regulator of mRNA Translation Promotes Extracellular Matrix Deposition to Accelerate Hepatocellular Carcinoma Initiation. 2023. https://doi.org/10.1101/2023.08.16.553544.
Grotehans, N.; McGarry, L.; Nolte, H.; Xavier, V.; Kroker, M.; Narbona-Pérez, Á. J.; Deshwal, S.; Giavalisco, P.; Langer, T.; MacVicar, T. Ribonucleotide Synthesis by NME6 Fuels Mitochondrial Gene Expression. EMBO J 2023, 42 (18), e113256. https://doi.org/10.15252/embj.2022113256.
Derby, S.; Dutton, L.; Strathdee, karen; Stevenson, K.; Clough, E.; Koessinger, A.; Yu, W.; Tian, Y.; Gilmour, L.; McGhee, E.; McGarrity-Cottrell, C.; Dibekeme, A. anderlinden; Collis, S.; Rominiyi, O.; Soares, L. L.; Peter; Solecki, G.; Winkler, F.; Carlin, L.; Inman, G.; Chalmers, A.; Norman, J.; Carruthers, R.; Birch, J. Inhibition of ATR Opposes Glioblastoma Invasion through Disruption of Cytoskeletal Networks and Integrin Internalisation via Macropinocytosis. 2023. https://doi.org/10.21203/rs.3.rs-967109/v2.
2022
Spiliopoulou, P.; Spear, S.; Mirza, H.; Garner, I.; McGarry, L.; Grundland-Freile, F.; Cheng, Z.; Ennis, D. P.; Iyer, N.; McNamara, S.; Natoli, M.; Mason, S.; Blyth, K.; Adams, P. D.; Roxburgh, P.; Fuchter, M. J.; Brown, B.; McNeish, I. A. Dual G9A/EZH2 Inhibition Stimulates Antitumor Immune Response in Ovarian High-Grade Serous Carcinoma. Mol Cancer Ther 2022, 21 (4), 522–534. https://doi.org/10.1158/1535-7163.mct-21-0743.
Schmidt, T.; Dabrowska, A.; Waldron, J.; Hodge, K.; Koulouras, G.; Gabrielsen, M.; Munro, J.; Tack, D.; Harris, G.; McGhee, E.; Scott, D.; Carlin, L.; Huang, D.; Le Quesne, J.; Zanivan, S.; Wilczynska, A.; Bushell, M. Purine-Rich RNA Sequences in the 5’UTR Site-Specifically Regulate eIF4A1-Unwinding through eIF4A1-Multimerisation to Facilitate Translation. 2022. https://doi.org/10.1101/2022.08.08.503179.
Sandilands, E.; Freckmann, E.; Román-Fernández, A.; McGarry, L.; Galbraith, L.; Mason, S.; Patel, R.; Anand, J.; Cartwright, J.; Leung, H.; Blyth, K.; Bryant, D. The Small GTPase ARF3 Controls Metastasis and Invasion Modality by Regulating N-Cadherin Levels. 2022. https://doi.org/10.1101/2022.04.25.489355.
Román-Fernández, A.; Mansour, M.; Kugeratski, F.; Anand, J.; Sandilands, E.; Galbraith, L.; Rakovic, K.; Freckmann, E.; Cumming, E.; Park, J.; Nikolatou, K.; Lilla, S.; Shaw, R.; Strachan, D.; Mason, S.; Patel, R.; McGarry, L.; Katoch, A.; Campbell, K.; Nixon, C.; Miller, C.; Leung, H.; Le Quesne, J.; Norman, J.; Zanivan, S.; Blyth, K.; Bryant, D. Spatial Regulation of the Glycocalyx Component Podocalyxin Is a Switch for Pro-Metastatic Function. 2022. https://doi.org/10.1101/2022.11.04.515043.
Nikolatou, K.; Sandilands, E.; Román-Fernández, A.; Cumming, E.; Freckmann, E.; Lilla, S.; Buetow, L.; McGarry, L.; Neilson, M.; Shaw, R.; Strachan, D.; Miller, C.; Huang, D.; McNeish, I.; Norman, J.; Zanivan, S.; Bryant, D. PTEN Deficiency Exposes a Requirement for an ARF GTPase Module in Integrin-Dependent Invasion in Ovarian Cancer. 2022. https://doi.org/10.1101/2022.11.29.518198.
Müller, M.; May, S.; Hall, H.; Kendall, T.; McGarry, L.; Blukacz, L.; Nuciforo, S.; Jamieson, T.; Phinichkusolchit, N.; Dhayade, S.; Leslie, J.; Sprangers, J.; Malviya, G.; Mrowinska, A.; Johnson, E.; McCain, M.; Halpin, J.; Kiourtis, C.; Georgakopoulou, A.; Nixon, C.; Clark, W.; Shaw, R.; Hedley, A.; Drake, T.; Tan, E. H.; Neilson, M.; Murphy, D.; Lewis, D.; Reeves, H.; Mann, D.; Blyth, K.; Heim, M.; Carlin, L.; Sansom, O.; Miller, C.; Bird, T. Human-Correlated Genetic HCC Models Identify Combination Therapy for Precision Medicine. 2022. https://doi.org/10.21203/rs.3.rs-1638504/v1.
Koessinger, A. L.; Cloix, C.; Koessinger, D.; Heiland, D. H.; Bock, F. J.; Strathdee, K.; Kinch, K.; Martínez-Escardó, L.; Paul, N. R.; Nixon, C.; Malviya, G.; Jackson, M. R.; Campbell, K. J.; Stevenson, K.; Davis, S.; Elmasry, Y.; Ahmed, A.; O’Prey, J.; Ichim, G.; Schnell, O.; Stewart, W.; Blyth, K.; Ryan, K. M.; Chalmers, A. J.; Norman, J. C.; Tait, S. W. G. Increased Apoptotic Sensitivity of Glioblastoma Enables Therapeutic Targeting by BH3-Mimetics. Cell Death Differ 2022, 29 (10), 2089–2104. https://doi.org/10.1038/s41418-022-01001-3.
Grotehans, N.; McGarry, L.; Nolte, H.; Kroker, M.; Narbona-Pérez, Á. J.; Deshwal, S.; Giavalisco, P.; Langer, T.; MacVicar, T. Ribonucleotide Synthesis by NME6 Fuels Mitochondrial Gene Expression. 2022. https://doi.org/10.1101/2022.11.29.518352.
Freckmann, E. C.; Sandilands, E.; Cumming, E.; Neilson, M.; Román-Fernández, A.; Nikolatou, K.; Nacke, M.; Lannagan, T. R. M.; Hedley, A.; Strachan, D.; Salji, M.; Morton, J. P.; McGarry, L.; Leung, H. Y.; Sansom, O. J.; Miller, C. J.; Bryant, D. M. Traject3d Allows Label-Free Identification of Distinct Co-Occurring Phenotypes within 3D Culture by Live Imaging. Nat Commun 2022, 13 (1), 5317. https://doi.org/10.1038/s41467-022-32958-x.
Buracco, S.; Singh, S.; Claydon, S.; Paschke, P.; Tweedy, L.; Whitelaw, J.; McGarry, L.; Thomason, P.; Insall, R. The Scar/WAVE Complex Drives Normal Actin Protrusions without the Arp2/3 Complex, but Proline-Rich Domains Are Required. 2022. https://doi.org/10.1101/2022.05.14.491902.
