Publications by authors named "Robert Insall"

117 Publications

Adhesion stimulates Scar/WAVE phosphorylation in mammalian cells.

Commun Integr Biol 2020 Dec 28;14(1):1-4. Epub 2020 Dec 28.

Institute of Cancer Sciences, University of Glasgow, Glasgow, UK.

The Scar/WAVE complex catalyzes the protrusion of pseudopods and lamellipods, and is therefore a principal regulator of cell migration. However, it is unclear how its activity is regulated, beyond a dependence on Rac. Phosphorylation of the proline-rich region, by kinases such as Erk2, has been suggested as an upstream activator. We have recently reported that phosphorylation is not required for complex activation. Rather, it occurs after Scar/WAVE has been activated, and acts as a modulator. Neither chemoattractant signaling nor Erk2 affects the amount of phosphorylation, though in Dictyostelium it is promoted by cell-substrate adhesion. We now report that cell-substrate adhesion also promotes Scar/WAVE2 phosphorylation in mammalian cells, suggesting that the process is evolutionarily conserved.
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http://dx.doi.org/10.1080/19420889.2020.1855854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781634PMC
December 2020

Fluorescence lifetime imaging with a megapixel SPAD camera and neural network lifetime estimation.

Sci Rep 2020 12 2;10(1):20986. Epub 2020 Dec 2.

Advanced Quantum Architecture Laboratory, Ecole Polytechnique Fédérale de Lausanne, 2002, Neuchâtel, Switzerland.

Fluorescence lifetime imaging microscopy (FLIM) is a key technology that provides direct insight into cell metabolism, cell dynamics and protein activity. However, determining the lifetimes of different fluorescent proteins requires the detection of a relatively large number of photons, hence slowing down total acquisition times. Moreover, there are many cases, for example in studies of cell collectives, where wide-field imaging is desired. We report scan-less wide-field FLIM based on a 0.5 MP resolution, time-gated Single Photon Avalanche Diode (SPAD) camera, with acquisition rates up to 1 Hz. Fluorescence lifetime estimation is performed via a pre-trained artificial neural network with 1000-fold improvement in processing times compared to standard least squares fitting techniques. We utilised our system to image HT1080-human fibrosarcoma cell line as well as Convallaria. The results show promise for real-time FLIM and a viable route towards multi-megapixel fluorescence lifetime images, with a proof-of-principle mosaic image shown with 3.6 MP.
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http://dx.doi.org/10.1038/s41598-020-77737-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710711PMC
December 2020

Structural Basis of CYRI-B Direct Competition with Scar/WAVE Complex for Rac1.

Structure 2021 Mar 19;29(3):226-237.e4. Epub 2020 Nov 19.

CRUK- Beatson Institute, Glasgow G61 1BD, UK; Institute of Cancer Sciences, University of Glasgow, Glasgow G61 1BD, UK; Department of Chemistry, KU Leuven, Celestijnenlaan 200G, 3001 Heverlee, Belgium. Electronic address:

Rac1 is a major regulator of actin dynamics, with GTP-bound Rac1 promoting actin assembly via the Scar/WAVE complex. CYRI competes with Scar/WAVE for interaction with Rac1 in a feedback loop regulating actin dynamics. Here, we reveal the nature of the CYRI-Rac1 interaction, through crystal structures of CYRI-B lacking the N-terminal helix (CYRI-BΔN) and the CYRI-BΔN:Rac1Q61L complex, providing the molecular basis for CYRI-B regulation of the Scar/WAVE complex. We reveal CYRI-B as having two subdomains - an N-terminal Rac1 binding subdomain with a unique Rac1-effector interface and a C-terminal Ratchet subdomain that undergoes conformational changes induced by Rac1 binding. Finally, we show that the CYRI protein family, CYRI-A and CYRI-B can produce an autoinhibited hetero- or homodimers, adding an additional layer of regulation to Rac1 signaling.
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http://dx.doi.org/10.1016/j.str.2020.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955166PMC
March 2021

Seeing around corners: Cells solve mazes and respond at a distance using attractant breakdown.

Science 2020 08;369(6507)

CRUK Beatson Institute, Switchback Road, Glasgow G61 1BD, UK.

During development and metastasis, cells migrate large distances through complex environments. Migration is often guided by chemotaxis, but simple chemoattractant gradients between a source and sink cannot direct cells over such ranges. We describe how self-generated gradients, created by cells locally degrading attractant, allow single cells to navigate long, tortuous paths and make accurate choices between live channels and dead ends. This allows cells to solve complex mazes efficiently. Cells' accuracy at finding live channels was determined by attractant diffusivity, cell speed, and path complexity. Manipulating these parameters directed cells in mathematically predictable ways; specific combinations can even actively misdirect them. We propose that the length and complexity of many long-range migratory processes, including inflammation and germ cell migration, means that self-generated gradients are needed for successful navigation.
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http://dx.doi.org/10.1126/science.aay9792DOI Listing
August 2020

Melanoblasts Populate the Mouse Choroid Earlier in Development Than Previously Described.

Invest Ophthalmol Vis Sci 2020 08;61(10):33

Liverpool Ocular Oncology Research Group, Department of Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom.

Purpose: Human choroidal melanocytes become evident in the last trimester of development, but very little is known about them. To better understand normal and diseased choroidal melanocyte biology we examined their precursors, melanoblasts (MB), in mouse eyes during development, particularly their relation to the developing vasculature and immune cells.

Methods: Naïve B6(Cg)-Tyrc-2J/J albino mice were used between embryonic (E) day 15.5 and postnatal (P) day 8, with adult controls. Whole eyes, posterior segments, or dissected choroidal wholemounts were stained with antibodies against tyrosinase-related protein 2, ionized calcium binding adaptor molecule-1 or isolectin B4, and examined by confocal microscopy. Immunoreactive cell numbers in the choroid were quantified with Imaris. One-way ANOVA with Tukey's post hoc test assessed statistical significance.

Results: Small numbers of MB were present in the presumptive choroid at E15.5 and E18.5. The density significantly increased between E18.5 (381.4 ± 45.8 cells/mm2) and P0 (695.2 ± 87.1 cells/mm2; P = 0.032). In postnatal eyes MB increased in density and formed multiple layers beneath the choriocapillaris. MB in the periocular mesenchyme preceded the appearance of vascular structures at E15.5. Myeloid cells (Ionized calcium binding adaptor molecule-1-positive) were also present at high densities from this time, and attained adult-equivalent densities by P8 (556.4 ± 73.6 cells/mm2).

Conclusions: We demonstrate that choroidal MB and myeloid cells are both present at very early stages of mouse eye development (E15.5). Although MB and vascularization seemed to be unlinked early in choroidal development, they were closely associated at later stages. MB did not migrate into the choroid in waves, nor did they have a consistent relationship with nerves.
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http://dx.doi.org/10.1167/iovs.61.10.33DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7441366PMC
August 2020

Cell-substrate adhesion drives Scar/WAVE activation and phosphorylation by a Ste20-family kinase, which controls pseudopod lifetime.

PLoS Biol 2020 08 3;18(8):e3000774. Epub 2020 Aug 3.

CRUK Beatson Institute, Glasgow, United Kingdom.

The Scar/WAVE complex is the principal catalyst of pseudopod and lamellipod formation. Here we show that Scar/WAVE's proline-rich domain is polyphosphorylated after the complex is activated. Blocking Scar/WAVE activation stops phosphorylation in both Dictyostelium and mammalian cells, implying that phosphorylation modulates pseudopods after they have been formed, rather than controlling whether they are initiated. Unexpectedly, phosphorylation is not promoted by chemotactic signaling but is greatly stimulated by cell:substrate adhesion and diminished when cells deadhere. Phosphorylation-deficient or phosphomimetic Scar/WAVE mutants are both normally functional and rescue the phenotype of knockout cells, demonstrating that phosphorylation is dispensable for activation and actin regulation. However, pseudopods and patches of phosphorylation-deficient Scar/WAVE last substantially longer in mutants, altering the dynamics and size of pseudopods and lamellipods and thus changing migration speed. Scar/WAVE phosphorylation does not require ERK2 in Dictyostelium or mammalian cells. However, the MAPKKK homologue SepA contributes substantially-sepA mutants have less steady-state phosphorylation, which does not increase in response to adhesion. The mutants also behave similarly to cells expressing phosphorylation-deficient Scar, with longer-lived pseudopods and patches of Scar recruitment. We conclude that pseudopod engagement with substratum is more important than extracellular signals at regulating Scar/WAVE's activity and that phosphorylation acts as a pseudopod timer by promoting Scar/WAVE turnover.
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http://dx.doi.org/10.1371/journal.pbio.3000774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7425996PMC
August 2020

Self-Generated Gradients Yield Exceptionally Robust Steering Cues.

Front Cell Dev Biol 2020 5;8:133. Epub 2020 Mar 5.

Cancer Research UK Beatson Institute, Glasgow, United Kingdom.

Chemotaxis is a widespread mechanism that allows migrating cells to steer to where they are needed. Attractant gradients may be imposed by external sources, or self-generated, when cells create their own steep local gradients by breaking down a prevalent, broadly distributed attractant. Here we show that chemotaxis works far more robustly toward self-generated gradients. Cells can only respond efficiently to a restricted range of attractant concentrations; if attractants are too dilute, their gradients are too shallow for cells to sense, but if they are too high, all receptors become saturated and cells cannot perceive spatial differences. Self-generated gradients are robust because cells maintain the attractant at optimal concentrations. A wave can recruit varying numbers of steered cells, and cells can take time to break down attractant before starting to migrate. Self-generated gradients can therefore operate over a greater range of attractant concentrations, larger distances, and longer times than imposed gradients. The robustness is further enhanced at low cell numbers if attractants also act as mitogens, and at high attractant concentrations if the enzymes that break down attractants are themselves induced by constant attractant levels.
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http://dx.doi.org/10.3389/fcell.2020.00133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7066204PMC
March 2020

Control of actin dynamics during cell motility.

F1000Res 2019 25;8. Epub 2019 Nov 25.

Institute of Cancer Sciences, University of Glasgow, Bearsden, G61 1BD, UK.

Actin polymerization is essential for cells to migrate, as well as for various cell biological processes such as cytokinesis and vesicle traffic. This brief review describes the mechanisms underlying its different roles and recent advances in our understanding. Actin usually requires "nuclei"-preformed actin filaments-to start polymerizing, but, once initiated, polymerization continues constitutively. The field therefore has a strong focus on nucleators, in particular the Arp2/3 complex and formins. These have different functions, are controlled by contrasting mechanisms, and generate alternate geometries of actin networks. The Arp2/3 complex functions only when activated by nucleation-promoting factors such as WASP, Scar/WAVE, WASH, and WHAMM and when binding to a pre-existing filament. Formins can be individually active but are usually autoinhibited. Each is controlled by different mechanisms and is involved in different biological roles. We also describe the processes leading to actin disassembly and their regulation and conclude with four questions whose answers are important for understanding actin dynamics but are currently unanswered.
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http://dx.doi.org/10.12688/f1000research.18669.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6880267PMC
January 2020

WASP Restricts Active Rac to Maintain Cells' Front-Rear Polarization.

Curr Biol 2019 12 27;29(24):4169-4182.e4. Epub 2019 Nov 27.

CRUK Beatson Institute, Switchback Road, Bearsden G61 1BD, UK; Institute of Cancer Sciences, University of Glasgow, University Avenue, Glasgow G12 8QQ, UK.

Efficient motility requires polarized cells, with pseudopods at the front and a retracting rear. Polarization is maintained by restricting the pseudopod catalyst, active Rac, to the front. Here, we show that the actin nucleation-promoting factor Wiskott-Aldrich syndrome protein (WASP) contributes to maintenance of front-rear polarity by controlling localization and cellular levels of active Rac. Dictyostelium cells lacking WASP inappropriately activate Rac at the rear, which affects their polarity and speed. WASP's Cdc42 and Rac interacting binding ("CRIB") motif has been thought to be essential for its activation. However, we show that the CRIB motif's biological role is unexpectedly complex. WASP CRIB mutants are no longer able to restrict Rac activity to the front, and cannot generate new pseudopods when SCAR/WAVE is absent. Overall levels of Rac activity also increase when WASP is unable to bind to Rac. However, WASP without a functional CRIB domain localizes normally at clathrin pits during endocytosis, and activates Arp2/3 complex. Similarly, chemical inhibition of Rac does not affect WASP localization or activation at sites of endocytosis. Thus, the interaction between small GTPases and WASP is more complex than previously thought-Rac regulates a subset of WASP functions, but WASP reciprocally restricts active Rac through its CRIB motif.
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http://dx.doi.org/10.1016/j.cub.2019.10.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926487PMC
December 2019

N-WASP Control of LPAR1 Trafficking Establishes Response to Self-Generated LPA Gradients to Promote Pancreatic Cancer Cell Metastasis.

Dev Cell 2019 11 24;51(4):431-445.e7. Epub 2019 Oct 24.

CRUK Beatson Institute, Glasgow G61 1BD, UK; Institute of Cancer Sciences, University of Glasgow, Glasgow G61 1BD, UK. Electronic address:

Pancreatic ductal adenocarcinoma is one of the most invasive and metastatic cancers and has a dismal 5-year survival rate. We show that N-WASP drives pancreatic cancer metastasis, with roles in both chemotaxis and matrix remodeling. lysophosphatidic acid, a signaling lipid abundant in blood and ascites fluid, is both a mitogen and chemoattractant for cancer cells. Pancreatic cancer cells break lysophosphatidic acid down as they respond to it, setting up a self-generated gradient driving tumor egress. N-WASP-depleted cells do not recognize lysophosphatidic acid gradients, leading to altered RhoA activation, decreased contractility and traction forces, and reduced metastasis. We describe a signaling loop whereby N-WASP and the endocytic adapter SNX18 promote lysophosphatidic acid-induced RhoA-mediated contractility and force generation by controlling lysophosphatidic acid receptor recycling and preventing degradation. This chemotactic loop drives collagen remodeling, tumor invasion, and metastasis and could be an important target against pancreatic cancer spread.
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http://dx.doi.org/10.1016/j.devcel.2019.09.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6863394PMC
November 2019

Analogies in 3D molecular visualisations: development of a cell biology animation 'How cells move - a new interpretation of old data'.

J Vis Commun Med 2020 Jan 23;43(1):35-46. Epub 2019 Oct 23.

The School of Simulation and Visualisation, Glasgow School of Art, Glasgow, UK.

Cell biology and imaging technology have vastly improved over the past decades, enabling scientists to dissect the inner workings of a cell. In addition to technical limits on spatial and temporal resolution, which obscure the picture at the molecular level, the sheer density and complexity of information impede clear understanding. 3D molecular visualisation has therefore blossomed as a way to translate molecular data in a more tangible form. Whilst the molecular machinery involved in cell locomotion has been extensively studied, existing narratives describing how cells generate the forces that drive movement remain unclear. Polymerisation of a protein called actin is clearly essential. The general belief in the cell migration field is that actin polymerisation's main role is to push the leading edge of the cell forwards, while the rest of the cell follows passively. The cell migration & chemotaxis group at the CRUK Beatson Institute propose an alternative hypothesis, in which actin filaments constitute cables. Motor proteins pull on these cables, causing them to behave like the treads of a tank and drive cell movement. This article describes the development of a 3D animation that uses analogical reasoning to contrast the 'tank' hypothesis for cell locomotion with the current dogma.
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http://dx.doi.org/10.1080/17453054.2019.1671814DOI Listing
January 2020

Screening by changes in stereotypical behavior during cell motility.

Sci Rep 2019 06 19;9(1):8784. Epub 2019 Jun 19.

Department of Life Sciences and Centre for Integrative Systems Biology and Bioinformatics, Imperial College, London, United Kingdom.

Stereotyped behaviors are series of postures that show very little variability between repeats. They have been used to classify the dynamics of individuals, groups and species without reference to the lower-level mechanisms that drive them. Stereotypes are easily identified in animals due to strong constraints on the number, shape, and relative positions of anatomical features, such as limbs, that may be used as landmarks for posture identification. In contrast, the identification of stereotypes in single cells poses a significant challenge as the cell lacks these landmark features, and finding constraints on cell shape is a non-trivial task. Here, we use the maximum caliber variational method to build a minimal model of cell behavior during migration. Without reference to biochemical details, we are able to make behavioral predictions over timescales of minutes using only changes in cell shape over timescales of seconds. We use drug treatment and genetics to demonstrate that maximum caliber descriptors can discriminate between healthy and aberrant migration, thereby showing potential applications for maximum caliber methods in automated disease screening, for example in the identification of behaviors associated with cancer metastasis.
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http://dx.doi.org/10.1038/s41598-019-45305-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6584642PMC
June 2019

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria.

J Vis Exp 2019 01 25(143). Epub 2019 Jan 25.

MRC Laboratory of Molecular Biology.

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.
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http://dx.doi.org/10.3791/58981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039707PMC
January 2019

PIKfyve/Fab1 is required for efficient V-ATPase and hydrolase delivery to phagosomes, phagosomal killing, and restriction of Legionella infection.

PLoS Pathog 2019 02 7;15(2):e1007551. Epub 2019 Feb 7.

Centre for Membrane Interactions and Dynamics, Department of Biomedical Sciences, University of Sheffield, Firth Court, Western Bank, Sheffield, United Kingdom.

By engulfing potentially harmful microbes, professional phagocytes are continually at risk from intracellular pathogens. To avoid becoming infected, the host must kill pathogens in the phagosome before they can escape or establish a survival niche. Here, we analyse the role of the phosphoinositide (PI) 5-kinase PIKfyve in phagosome maturation and killing, using the amoeba and model phagocyte Dictyostelium discoideum. PIKfyve plays important but poorly understood roles in vesicular trafficking by catalysing formation of the lipids phosphatidylinositol (3,5)-bisphosphate (PI(3,5)2) and phosphatidylinositol-5-phosphate (PI(5)P). Here we show that its activity is essential during early phagosome maturation in Dictyostelium. Disruption of PIKfyve inhibited delivery of both the vacuolar V-ATPase and proteases, dramatically reducing the ability of cells to acidify newly formed phagosomes and digest their contents. Consequently, PIKfyve- cells were unable to generate an effective antimicrobial environment and efficiently kill captured bacteria. Moreover, we demonstrate that cells lacking PIKfyve are more susceptible to infection by the intracellular pathogen Legionella pneumophila. We conclude that PIKfyve-catalysed phosphoinositide production plays a crucial and general role in ensuring early phagosomal maturation, protecting host cells from diverse pathogenic microbes.
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http://dx.doi.org/10.1371/journal.ppat.1007551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382210PMC
February 2019

Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo.

Curr Biol 2018 11 1;28(22):3674-3684.e6. Epub 2018 Nov 1.

Division of Molecular Cell Biology, Zoological Institute, Technische Universität Braunschweig, Spielmannstrasse 7, 38106 Braunschweig, Germany; Cell Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany. Electronic address:

Cell migration often involves the formation of sheet-like lamellipodia generated by branched actin filaments. The branches are initiated when Arp2/3 complex [1] is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family [2]. Recent structural studies defined two independent Rac binding sites on WRC within the Sra-1/PIR121 subunit of the pentameric WRC [3, 4], but the functions of these sites in vivo have remained unknown. Here we dissect the mechanism of WRC activation and the in vivo relevance of distinct Rac binding sites on Sra-1, using CRISPR/Cas9-mediated gene disruption of Sra-1 and its paralog PIR121 in murine B16-F1 cells combined with Sra-1 mutant rescue. We show that the A site, positioned adjacent to the binding region of WAVE-WCA mediating actin and Arp2/3 complex binding, is the main site for allosteric activation of WRC. In contrast, the D site toward the C terminus is dispensable for WRC activation but required for optimal lamellipodium morphology and function. These results were confirmed in evolutionarily distant Dictyostelium cells. Moreover, the phenotype seen in D site mutants was recapitulated in Rac1 E31 and F37 mutants; we conclude these residues are important for Rac-D site interaction. Finally, constitutively activated WRC was able to induce lamellipodia even after both Rac interaction sites were lost, showing that Rac interaction is not essential for membrane recruitment. Our data establish that physical interaction with Rac is required for WRC activation, in particular through the A site, but is not mandatory for WRC accumulation in the lamellipodium.
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http://dx.doi.org/10.1016/j.cub.2018.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264382PMC
November 2018

Fam49/CYRI interacts with Rac1 and locally suppresses protrusions.

Nat Cell Biol 2018 10 24;20(10):1159-1171. Epub 2018 Sep 24.

CRUK Beatson Institute, Glasgow, UK.

Actin-based protrusions are reinforced through positive feedback, but it is unclear what restricts their size, or limits positive signals when they retract or split. We identify an evolutionarily conserved regulator of actin-based protrusion: CYRI (CYFIP-related Rac interactor) also known as Fam49 (family of unknown function 49). CYRI binds activated Rac1 via a domain of unknown function (DUF1394) shared with CYFIP, defining DUF1394 as a Rac1-binding module. CYRI-depleted cells have broad lamellipodia enriched in Scar/WAVE, but reduced protrusion-retraction dynamics. Pseudopods induced by optogenetic Rac1 activation in CYRI-depleted cells are larger and longer lived. Conversely, CYRI overexpression suppresses recruitment of active Scar/WAVE to the cell edge, resulting in short-lived, unproductive protrusions. CYRI thus focuses protrusion signals and regulates pseudopod complexity by inhibiting Scar/WAVE-induced actin polymerization. It thus behaves like a 'local inhibitor' as predicted in widely accepted mathematical models, but not previously identified in cells. CYRI therefore regulates chemotaxis, cell migration and epithelial polarization by controlling the polarity and plasticity of protrusions.
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http://dx.doi.org/10.1038/s41556-018-0198-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6863750PMC
October 2018

Expression of N471D strumpellin leads to defects in the endolysosomal system.

Dis Model Mech 2018 09 13;11(9). Epub 2018 Sep 13.

Center for Biochemistry, Institute of Biochemistry I, Medical Faculty, University of Cologne, 50931 Cologne, Germany

Hereditary spastic paraplegias (HSPs) are genetically diverse and clinically characterised by lower limb weakness and spasticity. The N471D and several other point mutations of human strumpellin (Str; also known as WASHC5), a member of the Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex, have been shown to cause a form of HSP known as spastic paraplegia 8 (SPG8). To investigate the molecular functions of wild-type (WT) and N417D Str, we generated Str cells and ectopically expressed Str-GFP or Str-GFP in Str and WT cells. Overexpression of both proteins apparently caused a defect in cell division, as we observed a clear increase in multinucleate cells. Real-time PCR analyses revealed no transcriptional changes in WASH complex subunits in Str cells, but western blots showed a twofold decrease in the SWIP subunit. GFP-trap experiments in conjunction with mass-spectrometric analysis revealed many previously known, as well as new, Str-interacting proteins, and also proteins that no longer bind to Str At the cellular level, Str cells displayed defects in cell growth, phagocytosis, macropinocytosis, exocytosis and lysosomal function. Expression of Str-GFP in Str cells rescued all observed defects. In contrast, expression of Str-GFP could not rescue lysosome morphology and exocytosis of indigestible material. Our results underscore a key role for the WASH complex and its core subunit, Str, in the endolysosomal system, and highlight the fundamental importance of the Str N471 residue for maintaining lysosome morphology and dynamics. Our data indicate that the SPG8-causing N471D mutation leads to a partial loss of Str function in the endolysosomal system. This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/dmm.033449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177004PMC
September 2018

Rapid and efficient genetic engineering of both wild type and axenic strains of Dictyostelium discoideum.

PLoS One 2018 30;13(5):e0196809. Epub 2018 May 30.

MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.

Dictyostelium has a mature technology for molecular-genetic manipulation based around transfection using several different selectable markers, marker re-cycling, homologous recombination and insertional mutagenesis, all supported by a well-annotated genome. However this technology is optimized for mutant, axenic cells that, unlike non-axenic wild type, can grow in liquid medium. There is a pressing need for methods to manipulate wild type cells and ones with defects in macropinocytosis, neither of which can grow in liquid media. Here we present a panel of molecular genetic techniques based on the selection of Dictyostelium transfectants by growth on bacteria rather than liquid media. As well as extending the range of strains that can be manipulated, these techniques are faster than conventional methods, often giving usable numbers of transfected cells within a few days. The methods and plasmids described here allow efficient transfection with extrachromosomal vectors, as well as chromosomal integration at a 'safe haven' for relatively uniform cell-to-cell expression, efficient gene knock-in and knock-out and an inducible expression system. We have thus created a complete new system for the genetic manipulation of Dictyostelium cells that no longer requires cell feeding on liquid media.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0196809PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5976153PMC
July 2018

The trimeric coiled-coil HSBP1 protein promotes WASH complex assembly at centrosomes.

EMBO J 2018 07 29;37(13). Epub 2018 May 29.

Ecole Polytechnique, CNRS UMR7654, Université Paris-Saclay, Palaiseau, France

The Arp2/3 complex generates branched actin networks that exert pushing forces onto different cellular membranes. WASH complexes activate Arp2/3 complexes at the surface of endosomes and thereby fission transport intermediates containing endocytosed receptors, such as α5β1 integrins. How WASH complexes are assembled in the cell is unknown. Here, we identify the small coiled-coil protein HSBP1 as a factor that specifically promotes the assembly of a ternary complex composed of CCDC53, WASH, and FAM21 by dissociating the CCDC53 homotrimeric precursor. HSBP1 operates at the centrosome, which concentrates the building blocks. HSBP1 depletion in human cancer cell lines and in amoebae phenocopies WASH depletion, suggesting a critical role of the ternary WASH complex for WASH functions. HSBP1 is required for the development of focal adhesions and of cell polarity. These defects impair the migration and invasion of tumor cells. Overexpression of HSBP1 in breast tumors is associated with increased levels of WASH complexes and with poor prognosis for patients.
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http://dx.doi.org/10.15252/embj.201797706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028030PMC
July 2018

A G-protein-coupled chemoattractant receptor recognizes lipopolysaccharide for bacterial phagocytosis.

PLoS Biol 2018 05 25;16(5):e2005754. Epub 2018 May 25.

Chemotaxis Signal Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Disease, National Institutes of Health, Rockville, Maryland, United States of America.

Phagocytes locate microorganisms via chemotaxis and then consume them using phagocytosis. Dictyostelium amoebas are stereotypical phagocytes that prey on diverse bacteria using both processes. However, as typical phagocytic receptors, such as complement receptors or Fcγ receptors, have not been found in Dictyostelium, it remains mysterious how these cells recognize bacteria. Here, we show that a single G-protein-coupled receptor (GPCR), folic acid receptor 1 (fAR1), simultaneously recognizes the chemoattractant folate and the phagocytic cue lipopolysaccharide (LPS), a major component of bacterial surfaces. Cells lacking fAR1 or its cognate G-proteins are defective in chemotaxis toward folate and phagocytosis of Klebsiella aerogenes. Computational simulations combined with experiments show that responses associated with chemotaxis can also promote engulfment of particles coated with chemoattractants. Finally, the extracellular Venus-Flytrap (VFT) domain of fAR1 acts as the binding site for both folate and LPS. Thus, fAR1 represents a new member of the pattern recognition receptors (PRRs) and mediates signaling from both bacterial surfaces and diffusible chemoattractants to reorganize actin for chemotaxis and phagocytosis.
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http://dx.doi.org/10.1371/journal.pbio.2005754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5969738PMC
May 2018

The Chemoattractant Glorin Is Inactivated by Ester Cleavage during Early Multicellular Development of Polysphondylium pallidum.

ACS Chem Biol 2018 06 24;13(6):1506-1513. Epub 2018 May 24.

Pharmaceutical Biology, Institute of Pharmacy , Friedrich Schiller University , Jena , Germany.

Among the amoebozoan species capable of forming fruiting bodies, the dictyostelid social amoebae stand out since they form true multicellular organisms by means of single cell aggregation. Upon food depletion, cells migrate across gradients of extracellular signals initiated by cells in aggregation centers. The model species that is widely used to study multicellular development of social amoebae, Dictyostelium discoideum, uses cyclic adenosine monophosphate (cAMP) as a chemoattractant to coordinate aggregation. Molecular phylogeny studies suggested that social amoebae evolved in four major groups, of which groups 1 and 2 are paraphyletic to groups 3 and 4. During early development, intercellular communication with cAMP appears to be restricted to group 4 species. Cells of group 1 and 2 taxa do not respond chemotactically to extracellular cAMP and likely use a dipeptide chemoattractant known as glorin ( N-propionyl-γ-L-glutamyl-L-ornithin-δ-lactam-ethylester) to regulate aggregation. Directional migration of glorin-responsive cells requires the periodic breakdown of the chemoattractant. Here, we identified an extracellular enzymatic activity (glorinase) in the glorin-responsive group 2 taxon Polysphondylium pallidum leading to the inactivation of glorin. We determined the inactivation mechanism to proceed via hydrolytic ethyl ester cleavage of the γ-glutamyl moiety of glorin. Synthetic glorinamide, in which the ethyl ester group was substituted by an ethyl amide group, had glorin-like biological activity but was resistant to degradation by glorinase. Our observations pave the way for future investigations toward an ancient eukaryotic chemotaxis system.
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http://dx.doi.org/10.1021/acschembio.8b00046DOI Listing
June 2018

WASP family proteins and formins compete in pseudopod- and bleb-based migration.

J Cell Biol 2018 02 30;217(2):701-714. Epub 2017 Nov 30.

Cancer Research UK Beatson Institute, Glasgow, Scotland, UK

Actin pseudopods induced by SCAR/WAVE drive normal migration and chemotaxis in eukaryotic cells. Cells can also migrate using blebs, in which the edge is driven forward by hydrostatic pressure instead of actin. In , loss of SCAR is compensated by WASP moving to the leading edge to generate morphologically normal pseudopods. Here we use an inducible double knockout to show that cells lacking both SCAR and WASP are unable to grow, make pseudopods or, unexpectedly, migrate using blebs. Remarkably, amounts and dynamics of actin polymerization are normal. Pseudopods are replaced in double SCAR/WASP mutants by aberrant filopods, induced by the formin dDia2. Further disruption of the gene for dDia2 restores cells' ability to initiate blebs and thus migrate, though pseudopods are still lost. Triple knockout cells still contain near-normal F-actin levels. This work shows that SCAR, WASP, and dDia2 compete for actin. Loss of SCAR and WASP causes excessive dDia2 activity, maintaining F-actin levels but blocking pseudopod and bleb formation and migration.
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http://dx.doi.org/10.1083/jcb.201705160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5800805PMC
February 2018

LPP3 mediates self-generation of chemotactic LPA gradients by melanoma cells.

J Cell Sci 2017 Oct 4;130(20):3455-3466. Epub 2017 Sep 4.

Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, UK.

Melanoma cells steer out of tumours using self-generated lysophosphatidic acid (LPA) gradients. The cells break down LPA, which is present at high levels around the tumours, creating a dynamic gradient that is low in the tumour and high outside. They then migrate up this gradient, creating a complex and evolving outward chemotactic stimulus. Here, we introduce a new assay for self-generated chemotaxis, and show that raising LPA levels causes a delay in migration rather than loss of chemotactic efficiency. Knockdown of the lipid phosphatase LPP3 - but not of its homologues LPP1 or LPP2 - diminishes the cell's ability to break down LPA. This is specific for chemotactically active LPAs, such as the 18:1 and 20:4 species. Inhibition of autotaxin-mediated LPA production does not diminish outward chemotaxis, but loss of LPP3-mediated LPA breakdown blocks it. Similarly, in both 2D and 3D invasion assays, knockdown of LPP3 diminishes the ability of melanoma cells to invade. Our results demonstrate that LPP3 is the key enzyme in the breakdown of LPA by melanoma cells, and confirm the importance of attractant breakdown in LPA-mediated cell steering.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.207514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5665449PMC
October 2017

Mroh1, a lysosomal regulator localized by WASH-generated actin.

J Cell Sci 2017 05 19;130(10):1785-1795. Epub 2017 Apr 19.

Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK

The steps leading to constitutive exocytosis are poorly understood. In WASH complex mutants, exocytosis is blocked, so cells that take up fluorescent dextran from the medium retain it and remain fluorescent. Here, we establish a FACS-based method to select cells that retain fluorescent dextran, allowing identification of mutants with disrupted exocytosis. Screening a pool of random mutants identified members of the WASH complex, as expected, and multiple mutants in the conserved HEAT-repeat-containing protein Mroh1. In mutants, endosomes develop normally until the stage where lysosomes neutralize to postlysosomes, but thereafter the WASH complex is recycled inefficiently, and subsequent exocytosis is substantially delayed. Mroh1 protein localizes to lysosomes in mammalian and cells. In , it accumulates on lysosomes as they mature and is removed, together with the WASH complex, shortly before the postlysosomes are exocytosed. WASH-generated F-actin is required for correct subcellular localization; in WASH complex mutants, and immediately after latrunculin treatment, Mroh1 relocalizes from the cytoplasm to small vesicles. Thus, Mroh1 is involved in a late and hitherto undefined actin-dependent step in exocytosis.
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http://dx.doi.org/10.1242/jcs.197210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5450189PMC
May 2017

F-actin forms an extensive filamentous network required for material exchange and parasite maturation.

Elife 2017 03 21;6. Epub 2017 Mar 21.

Wellcome Centre for Molecular Parasitology, Institute of Infection, Immunity & Inflammation, University of Glasgow, Glasgow, United Kingdom.

Apicomplexan actin is important during the parasite's life cycle. Its polymerization kinetics are unusual, permitting only short, unstable F-actin filaments. It has not been possible to study actin in vivo and so its physiological roles have remained obscure, leading to models distinct from conventional actin behaviour. Here a modified version of the commercially available actin-chromobody was tested as a novel tool for visualising F-actin dynamics in Cb labels filamentous actin structures within the parasite cytosol and labels an extensive F-actin network that connects parasites within the parasitophorous vacuole and allows vesicles to be exchanged between parasites. In the absence of actin, parasites lack a residual body and inter-parasite connections and grow in an asynchronous and disorganized manner. Collectively, these data identify new roles for actin in the intracellular phase of the parasites lytic cycle and provide a robust new tool for imaging parasitic F-actin dynamics.
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http://dx.doi.org/10.7554/eLife.24119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5375643PMC
March 2017

Coordination by Cdc42 of Actin, Contractility, and Adhesion for Melanoblast Movement in Mouse Skin.

Curr Biol 2017 Mar 23;27(5):624-637. Epub 2017 Feb 23.

CRUK Beatson Institute, University of Glasgow, Switchback Road, Bearsden, Glasgow G61 1BD, UK. Electronic address:

The individual molecular pathways downstream of Cdc42, Rac, and Rho GTPases are well documented, but we know surprisingly little about how these pathways are coordinated when cells move in a complex environment in vivo. In the developing embryo, melanoblasts originating from the neural crest must traverse the dermis to reach the epidermis of the skin and hair follicles. We previously established that Rac1 signals via Scar/WAVE and Arp2/3 to effect pseudopod extension and migration of melanoblasts in skin. Here we show that RhoA is redundant in the melanocyte lineage but that Cdc42 coordinates multiple motility systems independent of Rac1. Similar to Rac1 knockouts, Cdc42 null mice displayed a severe loss of pigmentation, and melanoblasts showed cell-cycle progression, migration, and cytokinesis defects. However, unlike Rac1 knockouts, Cdc42 null melanoblasts were elongated and displayed large, bulky pseudopods with dynamic actin bursts. Despite assuming an elongated shape usually associated with fast mesenchymal motility, Cdc42 knockout melanoblasts migrated slowly and inefficiently in the epidermis, with nearly static pseudopods. Although much of the basic actin machinery was intact, Cdc42 null cells lacked the ability to polarize their Golgi and coordinate motility systems for efficient movement. Loss of Cdc42 de-coupled three main systems: actin assembly via the formin FMNL2 and Arp2/3, active myosin-II localization, and integrin-based adhesion dynamics.
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http://dx.doi.org/10.1016/j.cub.2017.01.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5344686PMC
March 2017

A plasma membrane template for macropinocytic cups.

Elife 2016 12 13;5. Epub 2016 Dec 13.

MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.

Macropinocytosis is a fundamental mechanism that allows cells to take up extracellular liquid into large vesicles. It critically depends on the formation of a ring of protrusive actin beneath the plasma membrane, which develops into the macropinocytic cup. We show that macropinocytic cups in are organised around coincident intense patches of PIP, active Ras and active Rac. These signalling patches are invariably associated with a ring of active SCAR/WAVE at their periphery, as are all examined structures based on PIP patches, including phagocytic cups and basal waves. Patch formation does not depend on the enclosing F-actin ring, and patches become enlarged when the RasGAP NF1 is mutated, showing that Ras plays an instructive role. New macropinocytic cups predominantly form by splitting from existing ones. We propose that cup-shaped plasma membrane structures form from self-organizing patches of active Ras/PIP, which recruit a ring of actin nucleators to their periphery.
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http://dx.doi.org/10.7554/eLife.20085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154761PMC
December 2016

Inference of the drivers of collective movement in two cell types: Dictyostelium and melanoma.

J R Soc Interface 2016 10;13(123)

School of Mathematics and Statistics, College of Science and Engineering, University of Glasgow, Glasgow, UK.

Collective cell movement is a key component of many important biological processes, including wound healing, the immune response and the spread of cancers. To understand and influence these movements, we need to be able to identify and quantify the contribution of their different underlying mechanisms. Here, we define a set of six candidate models-formulated as advection-diffusion-reaction partial differential equations-that incorporate a range of cell movement drivers. We fitted these models to movement assay data from two different cell types: Dictyostelium discoideum and human melanoma. Model comparison using widely applicable information criterion suggested that movement in both of our study systems was driven primarily by a self-generated gradient in the concentration of a depletable chemical in the cells' environment. For melanoma, there was also evidence that overcrowding influenced movement. These applications of model inference to determine the most likely drivers of cell movement indicate that such statistical techniques have potential to support targeted experimental work in increasing our understanding of collective cell movement in a range of systems.
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http://dx.doi.org/10.1098/rsif.2016.0695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5095226PMC
October 2016

WASH drives early recycling from macropinosomes and phagosomes to maintain surface phagocytic receptors.

Proc Natl Acad Sci U S A 2016 10 19;113(40):E5906-E5915. Epub 2016 Sep 19.

Department of Biomedical Sciences, Centre for Membrane Interactions and Dynamics, University of Sheffield, Sheffield S10 2TN, United Kingdom; Bateson Centre, University of Sheffield, Sheffield S10 2TN, United Kingdom;

Macropinocytosis is an ancient mechanism that allows cells to harvest nutrients from extracellular media, which also allows immune cells to sample antigens from their surroundings. During macropinosome formation, bulk plasma membrane is internalized with all its integral proteins. It is vital for cells to salvage these proteins before degradation, but the mechanisms for sorting them are not known. Here we describe the evolutionarily conserved recruitment of the WASH (WASP and SCAR homolog) complex to both macropinosomes and phagosomes within a minute of internalization. Using Dictyostelium, we demonstrate that WASH drives protein sorting and recycling from macropinosomes and is thus essential to maintain surface receptor levels and sustain phagocytosis. WASH functionally interacts with the retromer complex at both early and late phases of macropinosome maturation, but mediates recycling via retromer-dependent and -independent pathways. WASH mutants consequently have decreased membrane levels of integrins and other surface proteins. This study reveals an important pathway enabling cells to sustain macropinocytosis without bulk degradation of plasma membrane components.
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http://dx.doi.org/10.1073/pnas.1524532113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5056073PMC
October 2016

Loss of strumpellin in the melanocytic lineage impairs the WASH Complex but does not affect coat colour.

Pigment Cell Melanoma Res 2016 09;29(5):559-71

Cancer Research UK Beatson Institute, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.

The five-subunit WASH complex generates actin networks that participate in endocytic trafficking, migration and invasion in various cell types. Loss of one of the two subunits WASH or strumpellin in mice is lethal, but little is known about their role in mammals in vivo. We explored the role of strumpellin, which has previously been linked to hereditary spastic paraplegia, in the mouse melanocytic lineage. Strumpellin knockout in melanocytes revealed abnormal endocytic vesicle morphology but no impairment of migration in vitro or in vivo and no change in coat colour. Unexpectedly, WASH and filamentous actin could still localize to vesicles in the absence of strumpellin, although the shape and size of vesicles was altered. Blue native PAGE revealed the presence of two distinct WASH complexes, even in strumpellin knockout cells, revealing that the WASH complex can assemble and localize to endocytic compartments in cells in the absence of strumpellin.
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http://dx.doi.org/10.1111/pcmr.12506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5082549PMC
September 2016