Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells

Hodgson, Louis; Shen, Fuyuan; Hahn, Klaus M.

doi:10.1002/0471143030.cb1411s46

Cited by 95 publications

(130 citation statements)

References 26 publications

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“…FRET analysis was done according to a previously described protocol (56). Briefly, FRET images were analyzed by first subtracting background from each individual CFP and YFP FRET image (CFP excitation, YFP emission).…”

Section: Methodsmentioning

confidence: 99%

Synthetic spatially graded Rac activation drives cell polarization and movement

Lin

Holmes

Wang

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

101

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Migrating cells possess intracellular gradients of active Rho GTPases, which serve as central hubs in transducing signals from extracellular receptors to cytoskeletal and adhesive machinery. However, it is unknown whether shallow exogenously induced intracellular gradients of Rho GTPases are sufficient to drive cell polarity and motility. Here, we use microfluidic control to generate gradients of a small molecule and thereby directly induce linear gradients of active, endogenous Rac without activation of chemotactic receptors. Gradients as low as 15% were sufficient not only to trigger cell migration up the chemical gradient but to induce both cell polarization and repolarization. Cellular response times were inversely proportional to the steepness of Rac inducer gradient in agreement with a mathematical model, suggesting a function for chemoattractant gradient amplification upstream of Rac. Increases in activated Rac levels beyond a well-defined threshold augmented polarization and decreased sensitivity to the imposed gradient. The threshold was governed by initial cell polarity and PI3K activity, supporting a role for both in defining responsiveness to Rac activation. Our results reveal that Rac can serve as a starting point in defining cell polarity. Furthermore, our methodology may serve as a template to investigate processes regulated by intracellular signaling gradients.cell signaling | synthetic biology | chemotaxis | signal transduction

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Section: Methodsmentioning

confidence: 99%

Synthetic spatially graded Rac activation drives cell polarization and movement

Lin

Holmes

Wang

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

101

View full text Add to dashboard Cite

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“…Cells were then FACS sorted to tightly gate for nearidentical expression levels of the biosensor, equating to ,20% of the endogenous protein levels (Bravo-Cordero et al, 2011). In addition, these methods of producing biosensor stable cell lines in order to achieve the low but uniform expression levels are described in detail in the previous publications Hodgson et al, 2010;Machacek et al, 2009;Pertz et al, 2006).…”

Section: Cell Culture and Dna Transfectionmentioning

confidence: 99%

“…Live-cell imaging of RhoC and RhoA FLARE.sc biosensors was done as previously described (Bravo-Cordero et al, 2011) Hodgson et al, 2010;Machacek et al, 2009;Pertz et al, 2006). Briefly, MTLn3 cells stably incorporating RhoC or RhoA biosensor were starved in L15 medium (Invitrogen, Carlsbad, CA) supplemented with 0.35% BSA (starvation medium), for 3-4 hours.…”

Section: Live-cell Imaging Of Rhoc and Rhoa Biosensorsmentioning

confidence: 99%

“…Because the RhoA/C FLARE.sc biosensors are based on single-chain designs in which the FRET donor and acceptor halves are linked into a single molecule via an optimized linker, the local concentrations of the FRET donor and acceptor molecules are always equimolar (Hodgson et al, 2010;Pertz et al, 2006). Because of this specific feature, we are able to make the simplifying assumption that the ratio of FRET to the donor emission would be sufficient to describe the donor occupancy states (Zal and Gascoigne, 2004) and the resulting ratiometric data are useful for making relativistic comparisons of the GTPase-occupancy states.…”

Section: Live-cell Imaging Of Rhoc and Rhoa Biosensorsmentioning

confidence: 99%

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Spatial regulation of RhoC activity defines protrusion formation in migrating cells

Bravo‐Cordero

Sharma

Roh-Johnson

et al. 2013

Journal of Cell Science

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SummaryProtrusion formation is the first step that precedes cell movement of motile cells. Spatial control of actin polymerization is necessary to achieve directional protrusion during cell migration. Here we show that the spatial coordinators p190RhoGEF and p190RhoGAP regulate actin polymerization during leading edge protrusions by regulating the actin barbed end distribution and amplitude. The distribution of RhoC activity and proper balance of cofilin activation achieved by p190RhoGEF and p190RhoGAP determines the direction of final protrusive activity. These findings provide a new insight into the dynamic plasticity in the amplitude and distribution of barbed ends, which can be modulated by fine-tuning RhoC activity by upstream GEFs and GAPs for directed cell motility.

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“…Together, coordinated programs of RhoA, Rac1 and Cdc42 activation/inactivation play prominent roles in processes such as endocytosis/exocytosis, adhesion and motility, which may subsequently impact upon proliferation and death/survival. Recent advances in the development of activation-state sensitive fluorescent probes have allowed temporal and spatial analysis of Rho protein activation, which has added significantly to our appreciation of Rho regulation and function (Hodgson et al 2010). Much of the early research on Rho protein function relied upon over-expression of dominant-negative mutants that reduced affinity for GTP and constitutively-active mutants that reduced GTP hydrolysis; however, more refined analysis has become possible with the rise of RNAi and knockout methodologies (Heasman and Ridley 2008).…”

Section: Introductionmentioning

confidence: 99%