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Machesky, L. M.

Publications and source records attributed to Machesky, L. M..

10 recordsLinked to original sources

Direct targeting of the WAVE-regulatory complex and the actin cytoskeleton by the Anaplasma phagocytophilum effector, AnkA

The obligate intracellular tick-borne pathogen Anaplasma phagocytophilum is unusual in its tropism for neutrophils, within which it survives and replicates. Central to the ability of this bacterium to colonize this hostile niche are secreted effector proteins. One such effector protein, AnkA, interacts with multiple host ligands in both the cytoplasm and the nucleus, yet how these interactions promote infection is largely unknown. We discovered that AnkA directly binds both the WAVE Regulatory Complex (WRC), a signal-integration hub that regulates the formation of branched actin networks underpinning diverse cell functions, and to monomeric and filamentous actin. We determined that AnkA uses two forms of molecular mimicry, first by binding to the Abi1 component of the WRC via a WRC-Recruiting Abi-binding Peptide (WRAP), and second by binding to actin via a WH2-like Actin Binding Motif (ABM). Via these interactions, AnkA can exert multi-factorial influence on the actin cytoskeleton and signaling pathways converging on this host structure. AnkA and the WRC co-localise during bacterial internalization and during the late stages of infection, suggesting a central role in both host-cell colonization and bacterial dissemination. Our work reveals AnkA as the first example of a bacterial effector that directly targets the WRC. The co-occurrence of WRAP- and ABM-like motifs in other bacterial effector proteins suggests that this mechanism of actin cytoskeleton subversion may represent a conserved strategy used by other obligate intracellular bacteria.

microbiology↗

DishCam: an open-source 3D-printed darkfield imager for microbial colonies and small organisms on Petri dishes

Petri dishes are routinely photographed using handheld cameras under reflected light. While adequate for large, distinct colonies, this approach fails to capture individual organisms or colonies that are transparent, do not reflect light well, or have fine structures, and is unsuitable for high quality timelapses. Darkfield illumination provides superior contrast for organisms on agar surfaces, but existing solutions rely on improvised setups with inconsistent lighting, or on large and expensive benchtop instruments that cannot be housed inside incubators. We present DishCam, an open-source device consisting entirely of 3D-printed parts and inexpensive off-the-shelf electronics, controlled by a Raspberry Pi running custom open-source software. DishCam produces high-contrast darkfield images using angled LED illumination beneath a Petri dish. All components used are off-the-shelf or easily 3D-printed. The camera and lights can be fully controlled through Raspberry Pi using our software. We demonstrate the device across multiple biological systems: Klebsiella aerogenes colonies of differing densities, Trypanosoma brucei colonies doing social motility, Dictyostelium discoideum streaming aggregation, and Caenorhabditis elegans on feeding substrates. The total cost of the basic device is approximately {pound}166 or $222, with the upgraded configuration costing approximately {pound}413 or $552, which includes a high-quality telephoto lens, SSD storage and incubator-compatible cables.

microbiology↗

CYRI-B loss promotes enlarged mature focal adhesions and restricts microtubule and ERC1 access to the cell leading edge

CYRI proteins promote lamellipodial dynamics by opposing Rac1-mediated activation of the Scar/WAVE complex. This activity also supports resolution of macropinocytic cups, promoting internalisation of surface proteins, including integrins. Here, we show that CYRI-B also promotes focal adhesion maturation and dynamics. Focal adhesions in CYRI-B-depleted cells show accelerated maturation and become excessively large. We probed the composition of these enlarged focal adhesions, using a Bio-ID screen, with paxillin as bait. Our screen revealed changes in the adhesome suggesting early activation of stress fibre contraction and depletion of the integrin internalisation mediator ERC1. Lack of CYRI-B leads to more stable lamellipodia and accumulation of polymerised actin in stress fibres. This actin acts as a barrier to microtubule targeting for adhesion turnover. Thus, our studies reveal an important connection between lamellipodia dynamics controlled by CYRI-B and microtubule targeting of ERC1 to modulate adhesion maturation and turnover.

cell biology↗

CYRI-B mediated macropinocytosis drives metastasis via lysophosphatidic acid receptor uptake

Pancreatic ductal adenocarcinoma carries a dismal prognosis, with high rates of metastasis and few treatment options. Hyperactivation of KRAS in almost all tumours drives RAC1 activation, conferring enhanced migratory and proliferative capacity as well as macropinocytosis. Macropinocytosis is well understood as a nutrient scavenging mechanism, but little is known about its functions in trafficking of signaling receptors. We find that CYRI-B is highly expressed in pancreatic tumours in a mouse model of KRAS and p53- driven pancreatic cancer. Deletion of CYRI-B accelerates tumourigenesis, leading to enhanced ERK and JNK-induced proliferation in precancerous lesions, indicating a role as a buffer of RAC1 hyperactivation in early stages. However, as disease progresses, loss of CYRI-B inhibits metastasis. CYRI-B depleted tumour cells show reduced chemotactic responses to lysophosphatidic acid, a major driver of tumour spread, due to impaired macropinocytic uptake of LPAR1 receptor. Overall, we implicate CYRI-B as a mediator of growth and signaling in pancreatic cancer, providing new insights into pathways controlling metastasis.

cancer biology↗

Inactivation of histone chaperone HIRA unmasks a link between normal embryonic development of melanoblasts and maintenance of adult melanocyte stem cells

Histone chaperone HIRA is thought to play a role in both early development and aging, but little is known about connections between the two processes. Here, we explore this relationship using a lineage-specific knockout mouse model, TyrCre::Hirafl/fl, in which HIRA is deficient in the pigmentary system consisting of embryonic melanoblasts, postnatal melanocytes and melanocyte stem cells (McSCs). Hira knockout leads to reduced melanoblast numbers during embryogenesis, but wild type numbers of melanocytes at birth, normally functioning juvenile and young adult McSCs, and only a very mildly hypopigmented first hair coat. However, on closer analysis, Hira knockout melanocytic cells of newborn mice exhibit molecular markers characteristic of cell aging and proliferative deficits. As they age, TyrCre::Hirafl/fl mice display marked defects in McSC maintenance and premature hair graying. Importantly, these defects are only observed when HIRA is inactivated during embryogenesis, not post-natally. This genetic model illustrates how normal embryonic development lays the foundation for maintenance of adult tissue specific stem cells and so suppression of degenerative phenotypes of aging.

developmental biology↗

Collagen-VI expression is negatively mechanosensitive in pancreatic cancer cells and supports the metastatic niche

Pancreatic cancer is a deadly disease with high rates of metastasis, though how tumor cells establish metastatic lesions is not fully understood. A key feature of primary pancreatic tumors is extensive fibrosis due to deposition of extracellular matrix. While pancreatic cancer cells are programmed by stimuli derived from a stiff ECM, metastasis requires loss of attachment as well as adaptation to a softer microenvironment upon reaching distant sites. Growing evidence suggests that stiff ECM influences pancreatic cancer cell behaviour. Here we argue that this influence is reversible and that pancreatic cancer cells can be reprogrammed upon sensing of soft substrates. Through use of engineered polyacrylamide hydrogels with tuneable mechanical properties, we show that Collagen-VI is specifically upregulated on soft substrates, due to a lack of integrin engagement and low YAP1 activity. Collagen-VI supports migration in vitro and metastasis formation in vivo. Metastatic nodules formed by pancreatic cancer cells lacking Col6a1 expression, were characterised by stromal cell-derived collagen-VI deposition, suggesting that collagen-VI, either cancer or stroma derived, is an essential component of the metastatic niche. Summary StatementCollagen-VI is expressed by pancreatic tumors and metastases in a mechanosensitive way to promote niche colonisation.

cancer biology↗

3D matrix adhesion composition facilitates nuclear force coupling to drive invasive cell migration

Cell invasion and metastasis is a multi-step process, initialised through the acquisition of a migratory phenotype and the ability to move through differing and complex 3D extracellular environments. In this study we set out to identify the parameters required for invasive cell migration in 3D environments. Cells interact with the extracellular matrix via transmembrane-spanning integrin adhesion complexes, which are well characterised in cells plated on 2D surfaces, yet much less is known about them in cells embedded in 3D matrices. We establish a technique to determine the composition of cell matrix adhesion complexes of invasive breast cancer cells in 3D matrices and on 2D surfaces and we identify an interaction complex enriched in 3D adhesive sites required for 3D invasive migration. Depletion of {beta}-PIX-Myosin18A (Myo18A) abolishes cancer cell invasion, without negatively affecting matrix degradation, Rho GTPase signalling, or protrusion formation in collagen matrices. Instead, in a mechanism only seen in cells moving through 3D matrix, {beta}-PIX and Myo18A drive the polarised recruitment of non-muscle Myosin 2A (NM2A) to the tips of protrusions. This recruitment of NM2A is required for the creation of an NM2A-NM2B isoform gradient, which ranges from the protrusion to the nucleus. We observe a requirement for active force transmission to the nucleus during invasive migration that is needed to pull the nucleus forward. We postulate that the establishment of the NM2A-NM2B actomyosin gradient facilitates the coupling of cell-matrix interactions at the protrusive cell front with nuclear movement, enabling effective invasive migration and front-rear cell polarity.

cell biology↗

Seeing around corners: Cells create chemotactic gradients to solve mazes and respond to distant cues in complex environments.

Chemotaxis, in which cells steer using chemical gradients, drives fundamental biological processes like embryogenesis, metastasis and immune responses. Self-generated chemotaxis, where cells break down abundant attractants to create gradients, is an important but under-studied aspect of physiological navigation. Here we show that self-generated gradients allow cells to navigate arbitrarily complex paths and, remarkably, make accurate choices about pathways they have not yet encountered. This enables cells to solve microfluidic mazes, even with initially homogeneous environments and distant correct destinations. We combine computational models and experiments to understand how cells anticipate environmental features, and how decision accuracy is determined by path complexity, attractant diffusibility and cell speed. This permits mazes that are easy or hard for cells to resolve, despite similar appearances. Counterintuitively, slowly-diffusing attractants can generate a "mirage", making cells prefer dead ends over correct paths. In vivo environments resemble complex mazes, and only self-generated gradients realistically explain cell behaviour.

cell biology↗

WASP restricts active Rac to maintain cells’ front-rear polarisation

Efficient motility requires polarised cells, with anterior pseudopods and a retracting rear. This polarisation requires that the pseudopod catalyst Rac is restricted to the front. Here we show that the Arp2/3 complex regulator WASP is important for maintaining front-rear polarity, using a mechanism that limits where active Rac localises. Dictyostelium cells lacking WASP inappropriately activate Rac and SCAR/WAVE at their rears, leading to reduced cell speed. WASP facilitates the internalisation of clathrin-coated pits, and its Rac-binding CRIB motif is considered essential for its localisation and activity. However, WASP mutants with deleted CRIB domains, or harbouring a new mutation that prevents Rac binding, localise normally, recruit Arp2/3 complex, and drive actin polymerisation. Similarly, Rac inhibitors do not block WASP localisation or activation. Despite this, WASP CRIB mutants cannot restore polarisation of active Rac. Thus, WASPs interaction with Rac regulates Rac activity and cell polarity, but is dispensable for activating actin polymerization.

cell biology↗

CYRI (FAM49) proteins are local inhibitors of Scar/WAVE induced lamellipodia that bind directly to active Rac1

Actin-based protrusions driving cell migration are reinforced through positive feedback, but it is unclear how the cell restricts the eventual size of a protrusion or limits positive signals to cause splitting or retraction. We have identified an evolutionarily conserved regulator of the protrusion machinery, which we name CYRI (CYFIP-related Rac interacting) protein. CYRI shows sequence similarity to the Scar/WAVE complex member CYFIP in a Domain of Unknown Function, DUF1394. CYRI binds specifically to activated Rac1 via a common motif shared with CYFIP, establishing DUF1394 as a new Rac1 binding domain. CYRI-depleted cells have broad, Scar/WAVE-enriched lamellipodia and enhanced Rac1 signaling. Conversely, CYRI overexpression suppresses spreading and dramatically sharpens protrusions into unproductive needles. CYRI proteins use dynamic inhibition of Scar/WAVE induced actin to focus positive protrusion signals and regulate pseudopod complexity. CYRI behaves like a \"local inhibitor\" predicted and described in widely accepted mathematical models, but not previously identified in living cells.

cell biology↗