A rapid inducible RNA decay system reveals fast mRNA decay in P-bodies.

RNA decay is vital for regulating mRNA abundance and gene expression. Existing technologies lack the spatiotemporal precision or transcript specificity to capture the stochastic and transient decay process. We devise a general strategy to inducibly recruit protein factors to modulate target RNA metabolism. Specifically, we introduce a Rapid Inducible Decay of RNA (RIDR) technology to degrade target mRNAs within minutes. The fast and synchronous induction enables direct visualization of mRNA decay dynamics in cells. Applying RIDR to endogenous ACTB mRNA reveals rapid formation and dissolution of RNA granules in pre-existing P-bodies. Time-resolved RNA distribution measurements demonstrate rapid RNA decay inside P-bodies, which is further supported by knocking down P-body constituent proteins. Light and oxidative stress modulate P-body behavior, potentially reconciling the contradictory literature about P-body function. This study reveals compartmentalized RNA decay kinetics, establishing RIDR as a pivotal tool for exploring the spatiotemporal RNA metabolism in cells.

Non-catalytic role of phosphoinositide 3-kinase in mesenchymal cell migration through non-canonical induction of p85ß/AP2-mediated endocytosis.

Class IA phosphoinositide 3-kinase (PI3K) galvanizes fundamental cellular processes such as migration, proliferation, and differentiation. To enable these multifaceted roles, the catalytic subunit p110 utilizes the multi-domain, regulatory subunit p85 through its inter SH2 domain (iSH2). In cell migration, its product PI(3,4,5)P3 generates locomotive activity. While non-catalytic roles are also implicated, underlying mechanisms and their relationship to PI(3,4,5)P3 signaling remain elusive. Here, we report that a disordered region of iSH2 contains AP2 binding motifs which can trigger clathrin and dynamin-mediated endocytosis independent of PI3K catalytic activity. The AP2 binding motif mutants of p85 aberrantly accumulate at focal adhesions and increase both velocity and persistency in fibroblast migration. We thus propose the dual functionality of PI3K in the control of cell motility, catalytic and non-catalytic, arising distinctly from juxtaposed regions within iSH2.

Calcium influx promotes PLEKHG4B localization to cell-cell junctions and regulates the integrity of junctional actin filaments.

PLEKHG4B is a Cdc42-targeting guanine-nucleotide exchange factor implicated in forming epithelial cell-cell junctions. Here we explored the mechanism regulating PLEKHG4B localization. PLEKHG4B localized to the basal membrane in normal Ca2+ medium but accumulated at cell-cell junctions upon ionomycin treatment. Ionomycin-induced junctional localization of PLEKHG4B was suppressed upon disrupting its annexin-A2 (ANXA2)-binding ability. Thus, Ca2+ influx and ANXA2 binding are crucial for PLEKHG4B localization to cell-cell junctions. Treatments with low Ca2+ or BAPTA-AM (an intracellular Ca2+ chelator) suppressed PLEKHG4B localization to the basal membrane. Mutations of the phosphoinositide-binding motif in the pleckstrin homology (PH) domain of PLEKHG4B or masking of membrane phosphatidylinositol-4,5-biphosphate [PI(4,5)P2] suppressed PLEKHG4B localization to the basal membrane, indicating that basal membrane localization of PLEKHG4B requires suitable intracellular Ca2+ levels and PI(4,5)P2 binding of the PH domain. Activation of mechanosensitive ion channels (MSCs) promoted PLEKHG4B localization to cell-cell junctions, and their inhibition suppressed it. Moreover, similar to the PLEKHG4B knockdown phenotypes, inhibition of MSCs or treatment with BAPTA-AM disturbed the integrity of actin filaments at cell-cell junctions. Taken together, our results suggest that Ca2+ influx plays crucial roles in PLEKHG4B localization to cell-cell junctions and the integrity of junctional actin organization, with MSCs contributing to this process.

Synthetic control of actin polymerization and symmetry breaking in active protocells.

Non-linear biomolecular interactions on the membranes drive membrane remodeling that underlies fundamental biological processes including chemotaxis, cytokinesis, and endocytosis. The multitude of biomolecules, the redundancy in their interactions, and the importance of spatiotemporal context in membrane organization hampers understanding the physical principles governing membrane mechanics. A minimal, in vitro system that models the functional interactions between molecular signaling and membrane remodeling, while remaining faithful to cellular physiology and geometry is powerful yet remains unachieved. Here, inspired by the biophysical processes underpinning chemotaxis, we reconstituted externally-controlled actin polymerization inside giant unilamellar vesicles, guiding self-organization on the membrane. We show that applying undirected external chemical inputs to this system results in directed actin polymerization and membrane deformation that are uncorrelated with upstream biochemical cues, indicating symmetry breaking. A biophysical model of the dynamics and mechanics of both actin polymerization and membrane shape suggests that inhomogeneous distributions of actin generate membrane shape deformations in a non-linear fashion, a prediction consistent with experimental measurements and subsequent local perturbations. The active protocellular system demonstrates the interplay between actin dynamics and membrane shape in a symmetry breaking context that is relevant to chemotaxis and a suite of other biological processes.

ActuAtor, a Listeria-inspired molecular tool for physical manipulation of intracellular organizations through de novo actin polymerization.

Form and function are often interdependent throughout biology. Inside cells, mitochondria have particularly attracted attention since both their morphology and functionality are altered under pathophysiological conditions. However, directly assessing their causal relationship has been beyond reach due to the limitations of manipulating mitochondrial morphology in a physiologically relevant manner. By engineering a bacterial actin regulator, ActA, we developed tools termed "ActuAtor" that inducibly trigger actin polymerization at arbitrary subcellular locations. The ActuAtor-mediated actin polymerization drives striking deformation and/or movement of target organelles, including mitochondria, Golgi apparatus, and nucleus. Notably, ActuAtor operation also disperses non-membrane-bound entities such as stress granules. We then implemented ActuAtor in functional assays, uncovering the physically fragmented mitochondria being slightly more susceptible to degradation, while none of the organelle functions tested are morphology dependent. The modular and genetically encoded features of ActuAtor should enable its application in studies of the form-function interplay in various intracellular contexts.

A Rapid Inducible RNA Decay system reveals fast mRNA decay in P-bodies.

RNA decay plays a crucial role in regulating mRNA abundance and gene expression. Modulation of RNA degradation is imperative to investigate an RNA's function. However, information regarding where and how RNA decay occurs remains scarce, partially because existing technologies fail to initiate RNA decay with the spatiotemporal precision or transcript specificity required to capture this stochastic and transient process. Here, we devised a general method that employs inducible tethering of regulatory protein factors to target RNAs and modulate their metabolism. Specifically, we established a Rapid Inducible Decay of RNA (RIDR) technology to degrade target mRNA within minutes. The fast and synchronous induction enabled direct visualization of mRNA decay dynamics in cells with spatiotemporal precision previously unattainable. When applying RIDR to endogenous ACTB mRNA, we observed rapid formation and disappearance of RNA granules, which coincided with pre-existing processing bodies (P-bodies). We measured the time-resolved RNA distribution in P-bodies and cytoplasm after induction, and compared different models of P-body function. We determined that mRNAs rapidly decayed in P-bodies upon induction. Additionally, we validated the functional role of P-bodies by knocking down specific a P-body constituent protein and RNA degradation enzyme. This study determined compartmentalized RNA decay kinetics for the first time. Together, RIDR provides a valuable and generalizable tool to study the spatial and temporal RNA metabolism in cells.

Non-catalytic role of phosphoinositide 3-kinase in mesenchymal cell migration through non-canonical induction of p85ß/AP-2-mediated endocytosis.

Class IA phosphoinositide 3-kinase (PI3K) galvanizes fundamental cellular processes such as migration, proliferation, and differentiation. To enable multifaceted roles, the catalytic subunit p110 utilizes a multi-domain, regulatory subunit p85 through its inter SH2 domain (iSH2). In cell migration, their product PI(3,4,5)P3 generates locomotive activity. While non-catalytic roles are also implicated, underlying mechanisms and its relationship to PI(3,4,5)P3 signaling remain elusive. Here, we report that a disordered region of iSH2 contains previously uncharacterized AP-2 binding motifs which can trigger clathrin and dynamin-mediated endocytosis independent of PI3K catalytic activity. The AP-2 binding motif mutants of p85 aberrantly accumulate at focal adhesions and upregulate both velocity and persistency in fibroblast migration. We thus propose the dual functionality of PI3K in the control of cell motility, catalytic and non-catalytic, arising distinctly from juxtaposed regions within iSH2.

Non-catalytic role of phosphoinositide 3-kinase in mesenchymal cell migration through non-canonical induction of p85ß/AP-2-mediated endocytosis.

Class IA phosphoinositide 3-kinase (PI3K) galvanizes fundamental cellular processes such as migration, proliferation, and differentiation. To enable multifaceted roles, the catalytic subunit p110 utilizes a multidomain, regulatory subunit p85 through its inter SH2 domain (iSH2). In cell migration, their product PI(3,4,5)P3 generates locomotive activity. While non-catalytic roles are also implicated, underlying mechanisms and its relationship to PI(3,4,5)P3 signaling remain elusive. Here, we report that a disordered region of iSH2 contains previously uncharacterized AP-2 binding motifs which can trigger clathrin and dynamin-mediated endocytosis independent of PI3K catalytic activity. The AP-2 binding motif mutants of p85 aberrantly accumulate at focal adhesions and upregulate both velocity and persistency in fibroblast migration. We thus propose the dual functionality of PI3K in the control of cell motility, catalytic and non-catalytic, arising distinctly from juxtaposed regions within iSH2.

Non-catalytic allostery in α-TAT1 by a phospho-switch drives dynamic microtubule acetylation.

Spatiotemporally dynamic microtubule acetylation underlies diverse physiological and pathological events. Despite its ubiquity, the molecular mechanisms that regulate the sole microtubule acetylating agent, α-tubulin-N-acetyltransferase-1 (α-TAT1), remain obscure. Here, we report that dynamic intracellular localization of α-TAT1 along with its catalytic activity determines efficiency of microtubule acetylation. Specifically, we newly identified a conserved signal motif in the intrinsically disordered C-terminus of α-TAT1, consisting of three competing regulatory elements-nuclear export, nuclear import, and cytosolic retention. Their balance is tuned via phosphorylation by CDK1, PKA, and CK2, and dephosphorylation by PP2A. While the unphosphorylated form binds to importins and resides both in cytosol and nucleus, the phosphorylated form binds to specific 14-3-3 adapters and accumulates in the cytosol for maximal substrate access. Unlike other molecules with a similar phospho-regulated signal motif, α-TAT1 uniquely uses the nucleus as a hideout. This allosteric spatial regulation of α-TAT1 function may help uncover a spatiotemporal code of microtubule acetylation in normal and aberrant cell behavior.

Multiple ciliary localization signals control INPP5E ciliary targeting.

Primary cilia are sensory membrane protrusions whose dysfunction causes ciliopathies. INPP5E is a ciliary phosphoinositide phosphatase mutated in ciliopathies like Joubert syndrome. INPP5E regulates numerous ciliary functions, but how it accumulates in cilia remains poorly understood. Herein, we show INPP5E ciliary targeting requires its folded catalytic domain and is controlled by four conserved ciliary localization signals (CLSs): LLxPIR motif (CLS1), W383 (CLS2), FDRxLYL motif (CLS3) and CaaX box (CLS4). We answer two long-standing questions in the field. First, partial CLS1-CLS4 redundancy explains why CLS4 is dispensable for ciliary targeting. Second, the essential need for CLS2 clarifies why CLS3-CLS4 are together insufficient for ciliary accumulation. Furthermore, we reveal that some Joubert syndrome mutations perturb INPP5E ciliary targeting, and clarify how each CLS works: (i) CLS4 recruits PDE6D, RPGR and ARL13B, (ii) CLS2-CLS3 regulate association to TULP3, ARL13B, and CEP164, and (iii) CLS1 and CLS4 cooperate in ATG16L1 binding. Altogether, we shed light on the mechanisms of INPP5E ciliary targeting, revealing a complexity without known parallels among ciliary cargoes.

Defunctionalizing intracellular organelles such as mitochondria and peroxisomes with engineered phospholipase A/acyltransferases.

Organelles vitally achieve multifaceted functions to maintain cellular homeostasis. Genetic and pharmacological approaches to manipulate individual organelles are powerful in probing their physiological roles. However, many of them are either slow in action, limited to certain organelles, or rely on toxic agents. Here, we design a generalizable molecular tool utilizing phospholipase A/acyltransferases (PLAATs) for rapid defunctionalization of organelles via remodeling of the membrane phospholipids. In particular, we identify catalytically active PLAAT truncates with minimal unfavorable characteristics. Chemically-induced translocation of the optimized PLAAT to the mitochondria surface results in their rapid deformation in a phospholipase activity dependent manner, followed by loss of luminal proteins as well as dissipated membrane potential, thus invalidating the functionality. To demonstrate wide applicability, we then adapt the molecular tool in peroxisomes, and observe leakage of matrix-resident functional proteins. The technique is compatible with optogenetic control, viral delivery and operation in primary neuronal cultures. Due to such versatility, the PLAAT strategy should prove useful in studying organelle biology of diverse contexts.

Dynamin is primed at endocytic sites for ultrafast endocytosis.

Dynamin mediates fission of vesicles from the plasma membrane during endocytosis. Typically, dynamin is recruited from the cytosol to endocytic sites, requiring seconds to tens of seconds. However, ultrafast endocytosis in neurons internalizes vesicles as quickly as 50 ms during synaptic vesicle recycling. Here, we demonstrate that Dynamin 1 is pre-recruited to endocytic sites for ultrafast endocytosis. Specifically, Dynamin 1xA, a splice variant of Dynamin 1, interacts with Syndapin 1 to form molecular condensates on the plasma membrane. Single-particle tracking of Dynamin 1xA molecules confirms the liquid-like property of condensates in vivo. When Dynamin 1xA is mutated to disrupt its interaction with Syndapin 1, the condensates do not form, and consequently, ultrafast endocytosis slows down by 100-fold. Mechanistically, Syndapin 1 acts as an adaptor by binding the plasma membrane and stores Dynamin 1xA at endocytic sites. This cache bypasses the recruitment step and accelerates endocytosis at synapses.

PIP2 determines length and stability of primary cilia by balancing membrane turnovers.

Primary cilia are sensory organelles on many postmitotic cells. The ciliary membrane is continuous with the plasma membrane but differs in its phospholipid composition with phosphatidylinositol 4,5-bisposphate (PIP2) being much reduced toward the ciliary tip. In order to determine the functional significance of this difference, we used chemically induced protein dimerization to rapidly synthesize or degrade PIP2 selectively in the ciliary membrane. We observed ciliary fission when PIP2 was synthesized and a growing ciliary length when PIP2 was degraded. Ciliary fission required local actin polymerisation in the cilium, the Rho kinase Rac, aurora kinase A (AurkA) and histone deacetylase 6 (HDAC6). This pathway was previously described for ciliary disassembly before cell cycle re-entry. Activating ciliary receptors in the presence of dominant negative dynamin also increased ciliary PIP2, and the associated vesicle budding required ciliary PIP2. Finally, ciliary shortening resulting from constitutively increased ciliary PIP2 was mediated by the same actin - AurkA - HDAC6 pathway. Taken together, changes in ciliary PIP2 are a unifying point for ciliary membrane stability and turnover. Different stimuli increase ciliary PIP2 to secrete vesicles and reduce ciliary length by a common pathway. The paucity of PIP2 in the distal cilium therefore ensures ciliary stability.

Growth and site-specific organization of micron-scale biomolecular devices on living mammalian cells.

Mesoscale molecular assemblies on the cell surface, such as cilia and filopodia, integrate information, control transport and amplify signals. Designer cell-surface assemblies could control these cellular functions. Such assemblies could be constructed from synthetic components ex vivo, making it possible to form such structures using modern nanoscale self-assembly and fabrication techniques, and then oriented on the cell surface. Here we integrate synthetic devices, micron-scale DNA nanotubes, with mammalian cells by anchoring them by their ends to specific cell surface receptors. These filaments can measure shear stresses between 0-2 dyn/cm2, a regime important for cell signaling. Nanotubes can also grow while anchored to cells, thus acting as dynamic cell components. This approach to cell surface engineering, in which synthetic biomolecular assemblies are organized with existing cellular architecture, could make it possible to build new types of sensors, machines and scaffolds that can interface with, control and measure properties of cells.

Synthetic design of farnesyl-electrostatic peptides for development of a protein kinase A membrane translocation switch.

Molecular switches that respond to a biochemical stimulus in cells have proven utility as a foundation for developing molecular sensors and actuators that could be used to address important biological questions. Developing a molecular switch unfortunately remains difficult as it requires elaborate coordination of sensing and actuation mechanisms built into a single molecule. Here, we rationally designed a molecular switch that changes its subcellular localization in response to an intended stimulus such as an activator of protein kinase A (PKA). By arranging the sequence for Kemptide in tandem, we designed a farnesylated peptide whose localization can dramatically change upon phosphorylation by PKA. After testing a different valence number of Kemptide as well as modulating the linker sequence connecting them, we identified an efficient peptide switch that exhibited dynamic translocation between plasma membranes and internal endomembranes in a PKA activity dependent manner. Due to the modular design and small size, our PKA switch can have versatile utility in future studies as a platform for visualizing and perturbing signal transduction pathways, as well as for performing synthetic operations in cells.

A molecular trap inside microtubules probes luminal access by soluble proteins.

The uniquely hollow structure of microtubules (MTs) confers characteristic mechanical and biological properties. Although most regulatory processes take place at the outer surface, molecular events inside MTs, such as α-tubulin acetylation, also play a critical role. However, how regulatory proteins reach the site of action remains obscure. To assess luminal accessibility, we first identified luminally positioned residues of ß-tubulin that can be fused to a protein of interest. We then developed a chemically inducible technique with which cytosolic proteins can be rapidly trapped at the lumen of intact MTs in cells. A luminal trapping assay revealed that soluble proteins of moderate size can enter the lumen via diffusion through openings at the MT ends and sides. Additionally, proteins forming a complex with tubulins can be incorporated to the lumen through the plus ends. Our approach may not only illuminate this understudied territory, but may also help understand its roles in MT-mediated functions.

Metabolic Compartmentalization at the Leading Edge of Metastatic Cancer Cells.

Despite advances in targeted therapeutics and understanding in molecular mechanisms, metastasis remains a substantial obstacle for cancer treatment. Acquired genetic mutations and transcriptional changes can promote the spread of primary tumor cells to distant tissues. Additionally, recent studies have uncovered that metabolic reprogramming of cancer cells is tightly associated with cancer metastasis. However, whether intracellular metabolism is spatially and temporally regulated for cancer cell migration and invasion is understudied. In this review, we highlight the emergence of a concept, termed "membraneless metabolic compartmentalization," as one of the critical mechanisms that determines the metastatic capacity of cancer cells. In particular, we focus on the compartmentalization of purine nucleotide metabolism (e.g., ATP and GTP) at the leading edge of migrating cancer cells through the uniquely phase-separated microdomains where dynamic exchange of nucleotide metabolic enzymes takes place. We will discuss how future insights may usher in a novel class of therapeutics specifically targeting the metabolic compartmentalization that drives tumor metastasis.

Rational design and implementation of a chemically inducible heterotrimerization system.

Chemically inducible dimerization (CID) uses a small molecule to induce binding of two different proteins. CID tools such as the FK506-binding protein-FKBP-rapamycin-binding- (FKBP-FRB)-rapamycin system have been widely used to probe molecular events inside and outside cells. While various CID tools are available, chemically inducible trimerization (CIT) does not exist, due to inherent challenges in designing a chemical that simultaneously binds three proteins with high affinity and specificity. Here, we developed CIT by rationally splitting FRB and FKBP. Cellular and structural datasets showed efficient trimerization of split pairs of FRB or FKBP with full-length FKBP or FRB, respectively, by rapamycin. CIT rapidly induced tri-organellar junctions and perturbed intended membrane lipids exclusively at select membrane contact sites. By conferring one additional condition to what is achievable with CID, CIT expands the types of manipulation in single live cells to address cell biology questions otherwise intractable and engineer cell functions for future synthetic biology applications.

Rational Design of a Protein Kinase A Nuclear-cytosol Translocation Reporter.

Protein Kinase A (PKA) exists as a tetrameric holoenzyme which activates with increase of cAMP and plays an important role in many physiological processes including cardiac physiology, neuronal development, and adipocyte function. Although this kinase has been the subject of numerous biosensor designs, a single-fluorophore reporter that performs comparably to Förster resonance energy transfer (FRET) has not yet been reported. Here, we have used basic observations of electrostatic interactions in PKA substrate recognition mechanism and nucleus localization sequence motif to design a phosphorylation switch that shuttles between the cytosol and the nucleus, a strategy that should be generalizable to all basophilic kinases. The resulting reporter yielded comparable kinetics and dynamic range to the PKA FRET reporter, AKAR3EV. We also performed basic characterization and demonstrated its potential use in monitoring multiple signaling molecules inside cells using basic fluorescence microscopy. Due to the single-fluorophore nature of this reporter, we envision that this could find broad applications in studies involving single cell analysis of PKA activity.