A Photocleavable Rapamycin Conjugate for Spatiotemporal Control of Small GTPase Activity

Umeda, Nobuhiro; Ueno, Takahiro; Pohlmeyer, Christopher W.; Nagano, Tetsuo; Inoue, Takanari

doi:10.1021/ja108258d

Cited by 125 publications

(99 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Dimerization is virtually irreversible on the time-scale of lipid signaling, and it requires a cofactor (rapamycin or a rapalogue) that must penetrate the cell membrane, thus affecting speed of action and limiting its applicability to living organisms. Moreover, drugs cannot be applied with subcellular precision, although the use of caged rapamycin activated by UV has been reported (14).…”

mentioning

confidence: 99%

Optogenetic control of phosphoinositide metabolism

Idevall‐Hagren

Dickson

Hille

et al. 2012

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

281

291

View full text Add to dashboard Cite

Phosphoinositides (PIs) are lipid components of cell membranes that regulate a wide variety of cellular functions. Here we exploited the blue light-induced dimerization between two plant proteins, cryptochrome 2 (CRY2) and the transcription factor CIBN, to control plasma membrane PI levels rapidly, locally, and reversibly. The inositol 5-phosphatase domain of OCRL (5-ptase OCRL ), which acts on PI(4,5)P 2 and PI(3,4,5)P 3 , was fused to the photolyase homology region domain of CRY2, and the CRY2-binding domain, CIBN, was fused to plasma membrane-targeting motifs. Blue-light illumination (458-488 nm) of mammalian cells expressing these constructs resulted in nearly instantaneous recruitment of 5-ptase OCRL to the plasma membrane, where it caused rapid (within seconds) and reversible (within minutes) dephosphorylation of its targets as revealed by diverse cellular assays: dissociation of PI(4,5)P 2 and PI (3,4,5)P 3 biosensors, disappearance of endocytic clathrin-coated pits, nearly complete inhibition of KCNQ2/3 channel currents, and loss of membrane ruffling. Focal illumination resulted in local and transient 5-ptase OCRL recruitment and PI(4,5)P 2 dephosphorylation, causing not only local collapse and retraction of the cell edge or process but also compensatory accumulation of the PI(4,5)P 2 biosensor and membrane ruffling at the opposite side of the cells. Using the same approach for the recruitment of PI3K, local PI(3,4,5)P 3 synthesis and membrane ruffling could be induced, with corresponding loss of ruffling distally to the illuminated region. This technique provides a powerful tool for dissecting with high spatial-temporal kinetics the cellular functions of various PIs and reversibly controlling the functions of downstream effectors of these signaling lipids.endocytosis | polarity | rapamycin | ion channel | ruffles P hosphoinositides (PIs) are key signaling components of cell membranes that result from the reversible phosphorylation of phosphatidylinositol at the 3, 4, and 5 positions of the inositol ring. These reactions give rise to seven PI species that regulate a variety of cell processes including membrane trafficking, cytoskeleton dynamics, cell migration, cytokinesis, and ion and metabolite fluxes across membranes. Their signaling is linked to their different subcellular localization, rapid turnover, and distinct repertoire of binding proteins (1-3). Given these properties, functional interrogation of PI signaling through experimental manipulation requires precise spatial and temporal control.To date, most studies investigating the role of these lipids have relied on pharmacological or genetic perturbations of the enzymes responsible for PI synthesis and degradation. Small molecules that affect these enzymes have been developed, but, as is the case with all drugs, their off-target effects cannot be excluded. Genetic manipulations (knockout, knockdown, and overexpression studies), as well as studies of patients harboring mutations in such enzymes, have greatly advanced our understanding of the funct...

show abstract

mentioning

confidence: 99%

Optogenetic control of phosphoinositide metabolism

Idevall‐Hagren

Dickson

Hille

et al. 2012

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

281

291

View full text Add to dashboard Cite

show abstract

“…Localization and signaling motifs can be linked to either domain, allowing spatial-temporal control of protein function. We used our combined system to study the effects of a rapidly induced intracellular gradient of activated Rac, which is an important regulator of cell polarity (27) and has been shown previously to induce migration when locally activated (9,(12)(13)(14)(15)(16).…”

mentioning

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.

Self Cite

101

View full text Add to dashboard Cite

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

show abstract

“…Of note, a photocleavable rapamycin analog was recently created to dimerize FKBP and FKBP12-rapamycin-associated protein (FRAP). In this case, the photocleavage was used to activate the rapamycin analog for spatial and temporal activation of the signaling event (22,23). We propose that the PhAP presented herein, in conjunction with a molecular trap expressed by a tissue-specific promoter, will not only afford spatial and temporal activation of a biological process in an animal model (e.g.…”

Section: Discussionmentioning

confidence: 99%

Photocleavable Dimerizer for the Rapid Reversal of Molecular Trap Antagonists

Ahmed

Xie

Horne

et al. 2014

Journal of Biological Chemistry

View full text Add to dashboard Cite

Developing a molecular tool that can rapidly turn on and off an acute antagonist will be immensely helpful to understand the initiation of a cellular program. Herein, we report the development of a photocleavable analog of AP20187, a cell permeable molecule used to dimerize FKBP fusion proteins and initiate biological signaling cascades, gene expression or disrupt protein‐protein interactions. We demonstrate that this reagent in conjugation with a molecular trap permits the ability to rapidly and specifically antagonize a molecular interaction in vitro and follow a biological process due to this acute antagonism (e.g., endosome dispersion). More importantly, photocleavage of the analog permits the unique ability to follow the cell’s return to homeostasis. Herein, we demonstrated the fine temporal control of endosome dispersion and restoration using an LC8 trap. We posit that this photocleavable AP20187 analog can be used in other systems where the dimerization of FKBP has been used to initiate signaling pathways (e.g., kinases). In toto, the combination of a molecular trap and the photocleavable analog opens up new avenues to study signaling events within a single cell in a whole organism. Grant Funding Source: Supported by the generous support of R21NS071166 and also by P30 CA033572 from the NCI

show abstract

A Photocleavable Rapamycin Conjugate for Spatiotemporal Control of Small GTPase Activity

Cited by 125 publications

References 23 publications

Optogenetic control of phosphoinositide metabolism

Optogenetic control of phosphoinositide metabolism

Synthetic spatially graded Rac activation drives cell polarization and movement

Photocleavable Dimerizer for the Rapid Reversal of Molecular Trap Antagonists

Contact Info

Product

Resources

About