Rapamycin-induced Heterodimerization Research Articles

Conditional Gene Deletion in Mammalian and Mosquito Stages of Plasmodium berghei Using Dimerizable Cre Recombinase.

Genome editing in the malaria parasite Plasmodium relies on homologous recombination and requires parasite transfection in asexual blood stages. Therefore, conditional genetic approaches are needed to delete genes that are essential during blood stage replication. Among these, the dimerizable Cre (DiCre) recombinase system has emerged as a powerful approach for conditional gene knockout in Plasmodium parasites. In this system, the Cre recombinase is expressed in the form of two separate, enzymatically inactive polypeptides. Rapamycin-induced heterodimerization of the two components restores recombinase activity, leading to site-specific excision of floxed DNA sequences. Here, we describe methods to generate genetically modified DiCre-expressing Plasmodium berghei mutants by introducing Lox sites upstream and downstream of a gene of interest and to induce conditional excision of the floxed gene in different stages of the parasite life cycle. Administration of rapamycin to P. berghei-infected mice allows conditional gene deletion in the asexual erythrocytic stages. Rapamycin-induced gene excision can also be achieved in P. berghei sexual blood stages prior to transmission to mosquitoes, or during sporogony by treating P. berghei-infected mosquitoes, both methods allowing functional studies in P. berghei mosquito stages. Finally, rapamycin can be administered to in vitro cell cultures in order to induce gene excision in P. berghei liver stages. Subsequent phenotyping allows for the analysis of essential gene function across the parasite life cycle stages.

The dimerisable Cre recombinase allows conditional genome editing in the mosquito stages of Plasmodium berghei.

Asexual blood stages of the malaria parasite are readily amenable to genetic modification via homologous recombination, allowing functional studies of parasite genes that are not essential in this part of the life cycle. However, conventional reverse genetics cannot be applied for the functional analysis of genes that are essential during asexual blood-stage replication. Various strategies have been developed for conditional mutagenesis of Plasmodium, including recombinase-based gene deletion, regulatable promoters, and mRNA or protein destabilization systems. Among these, the dimerisable Cre (DiCre) recombinase system has emerged as a powerful approach for conditional gene deletion in P. falciparum. In this system, the bacteriophage Cre is expressed in the form of two separate, enzymatically inactive polypeptides, each fused to a different rapamycin-binding protein. Rapamycin-induced heterodimerization of the two components restores recombinase activity. We have implemented the DiCre system in the rodent malaria parasite P. berghei, and show that rapamycin-induced excision of floxed DNA sequences can be achieved with very high efficiency in both mammalian and mosquito parasite stages. This tool can be used to investigate the function of essential genes not only in asexual blood stages, but also in other parts of the malaria parasite life cycle.

The ER-Localized Transmembrane Protein TMEM39A/SUSR2 Regulates Autophagy by Controlling the Trafficking of the PtdIns(4)P Phosphatase SAC1.

TMEM39A, encoding an ER-localized transmembrane protein, is a susceptibility locus for multiple autoimmune diseases. The molecular function of TMEM39A remains completely unknown. Here we demonstrated that TMEM39A, also called SUSR2, modulates autophagy activity by regulating the spatial distribution and levels of PtdIns(4)P. Depletion of SUSR2 elevates late endosomal/lysosomal PtdIns(4)P levels, facilitating recruitment of the HOPS complex to promote assembly of the SNARE complex for autophagosome maturation. SUSR2 knockdown also increases the degradative capability of lysosomes. Mechanistically, SUSR2 interacts with the ER-localized PtdIns(4)P phosphatase SAC1 and also the COPII SEC23/SEC24 subunits to promote the ER-to-Golgi transport of SAC1. Retention of SAC1 on the ER in SUSR2 knockdown cells increases the level of PtdIns(3)P produced by the VPS34 complex, promoting autophagosome formation. Our study reveals that TMEM39A/SUSR2 acts as an adaptor protein for efficient export of SAC1from the ER and provides insights into the pathogenesis of diseases associated with TMEM39A mutations.

Label-free Single-Molecule Quantification of Rapamycin-induced FKBP-FRB Dimerization for Direct Control of Cellular Mechanotransduction.

Chemically induced dimerization (CID) has been applied to study numerous biological processes and has important pharmacological applications. However, the complex multistep interactions under various physical constraints involved in CID impose a great challenge for the quantification of the interactions. Furthermore, the mechanical stability of the ternary complexes has not been characterized; hence, their potential application in mechanotransduction studies remains unclear. Here, we report a single-molecule detector that can accurately quantify almost all key interactions involved in CID and the mechanical stability of the ternary complex, in a label-free manner. Its application is demonstrated using rapamycin-induced heterodimerization of FRB and FKBP as an example. We revealed the sufficient mechanical stability of the FKBP/rapamycin/FRB ternary complex and demonstrated its utility in the precise switching of talin-mediated force transmission in integrin-based cell adhesions.

FerriTag is a new genetically-encoded inducible tag for correlative light-electron microscopy

A current challenge is to develop tags to precisely visualize proteins in cells by light and electron microscopy. Here, we introduce FerriTag, a genetically-encoded chemically-inducible tag for correlative light-electron microscopy. FerriTag is a fluorescent recombinant electron-dense ferritin particle that can be attached to a protein-of-interest using rapamycin-induced heterodimerization. We demonstrate the utility of FerriTag for correlative light-electron microscopy by labeling proteins associated with various intracellular structures including mitochondria, plasma membrane, and clathrin-coated pits and vesicles. FerriTagging has a good signal-to-noise ratio and a labeling resolution of approximately 10 nm. We demonstrate how FerriTagging allows nanoscale mapping of protein location relative to a subcellular structure, and use it to detail the distribution and conformation of huntingtin-interacting protein 1 related (HIP1R) in and around clathrin-coated pits.

Carboxyl Terminus as a Key Regulator of Gating and Signaling of L-Type Calcium Channels

L-type calcium channels (CaV1) play vital roles in critical biological processes including calcium dynamics and gene regulation, where the carboxyl terminus of CaV1 gets closely involved. In this presentation, we are introducing some of the recent progress and findings about C-tail's roles in CaV1 gating and signaling. First, we report C-terminus mediated inhibition (CMI) for CaV1.3 that multiple motifs coordinate to compete calmodulin (CaM) off the channel, and tune down Ca2+ current and Ca2+ influx toward the lower limits determined by end-stage CDI (Ca2+-dependent inactivation). Acute CMI by rapamycin-inducible heterodimerization helps reconcile the concurrent activation/inactivation attenuations to ensure CMI as a new modality of channel inhibition (Liu N, et al., eLife, 2017). Data suggest that CMI could also profoundly affect the downstream Ca2+ signaling especially the CaV1-dependent gene regulation. Such importance of C-tail is in part demonstrated by the work dealing with the side-effects of CaM-based GCaMP, one of the most popular types of Ca2+ probes. We unveil the intrinsic problem prevailing over different versions and applications, that GCaMP acts as impaired CaM of apo and Ca2+ forms both critical to C-tail functions in CaV1, resulting into distorted Ca2+ dynamics and altered gene expression. We then design and implement GCaMP-X, which successfully resolves the problems of detrimental nuclear accumulation, acute and chronic Ca2+ dysregulation, and abnormal cell morphogenesis. The interplays between the C-terminus and signaling proteins well demonstrate the ultimate and synergistic tasks of CaV1 complex: to deliver Ca2+ of appropriate spatiotemporal patterns (gating) to the right listeners and actuators (signaling).

Cooperative and acute inhibition by multiple C-terminal motifs of L-type Ca2+ channels.

Inhibitions and antagonists of L-type Ca2+ channels are important to both research and therapeutics. Here, we report C-terminus mediated inhibition (CMI) for CaV1.3 that multiple motifs coordinate to tune down Ca2+ current and Ca2+ influx toward the lower limits determined by end-stage CDI (Ca2+-dependent inactivation). Among IQV (preIQ3-IQ domain), PCRD and DCRD (proximal or distal C-terminal regulatory domain), spatial closeness of any two modules, e.g., by constitutive fusion, facilitates the trio to form the complex, compete against calmodulin, and alter the gating. Acute CMI by rapamycin-inducible heterodimerization helps reconcile the concurrent activation/inactivation attenuations to ensure Ca2+ influx is reduced, in that Ca2+ current activated by depolarization is potently (~65%) inhibited at the peak (full activation), but not later on (end-stage inactivation, ~300 ms). Meanwhile, CMI provides a new paradigm to develop CaV1 inhibitors, the therapeutic potential of which is implied by computational modeling of CaV1.3 dysregulations related to Parkinson's disease.

Locked and proteolysis-based transcription activator-like effector (TALE) regulation

Development of orthogonal, designable and adjustable transcriptional regulators is an important goal of synthetic biology. Their activity has been typically modulated through stimulus-induced oligomerization or interaction between the DNA-binding and activation/repression domain. We exploited a feature of the designable Transcription activator-like effector (TALE) DNA-binding domain that it winds around the DNA which allows to topologically prevent it from binding by intramolecular cyclization. This new approach was investigated through noncovalent ligand-induced cyclization or through a covalent split intein cyclization strategy, where the topological inhibition of DNA binding by cyclization and its restoration by a proteolytic release of the topologic constraint was expected. We show that locked TALEs indeed have diminished DNA binding and regain full transcriptional activity by stimulation with the rapamycin ligand or site-specific proteolysis of the peptide linker, with much higher level of activation than rapamycin-induced heterodimerization. Additionally, we demonstrated reversibility, activation of genomic targets and implemented logic gates based on combinations of protein cyclization, proteolytic cleavage and ligand-induced dimerization, where the strongest fold induction was achieved by the proteolytic cleavage of a repression domain from a linear TALE.

Improved Methodical Approach for Quantitative BRET Analysis of G Protein Coupled Receptor Dimerization

G Protein Coupled Receptors (GPCR) can form dimers or higher ordered oligomers, the process of which can remarkably influence the physiological and pharmacological function of these receptors. Quantitative Bioluminescence Resonance Energy Transfer (qBRET) measurements are the gold standards to prove the direct physical interaction between the protomers of presumed GPCR dimers. For the correct interpretation of these experiments, the expression of the energy donor Renilla luciferase labeled receptor has to be maintained constant, which is hard to achieve in expression systems. To analyze the effects of non-constant donor expression on qBRET curves, we performed Monte Carlo simulations. Our results show that the decrease of donor expression can lead to saturation qBRET curves even if the interaction between donor and acceptor labeled receptors is non-specific leading to false interpretation of the dimerization state. We suggest here a new approach to the analysis of qBRET data, when the BRET ratio is plotted as a function of the acceptor labeled receptor expression at various donor receptor expression levels. With this method, we were able to distinguish between dimerization and non-specific interaction when the results of classical qBRET experiments were ambiguous. The simulation results were confirmed experimentally using rapamycin inducible heterodimerization system. We used this new method to investigate the dimerization of various GPCRs, and our data have confirmed the homodimerization of V2 vasopressin and CaSR calcium sensing receptors, whereas our data argue against the heterodimerization of these receptors with other studied GPCRs, including type I and II angiotensin, β2 adrenergic and CB1 cannabinoid receptors.

Conditional targeting of MAD1 to kinetochores is sufficient to reactivate the spindle assembly checkpoint in metaphase

Fidelity of chromosome segregation is monitored by the spindle assembly checkpoint (SAC). Key components of the SAC include MAD1, MAD2, BUB1, BUB3, BUBR1, and MPS1. These proteins accumulate on kinetochores in early prometaphase but are displaced when chromosomes attach to microtubules and/or biorient on the mitotic spindle. As a result, stable attachment of the final chromosome satisfies the SAC, permitting activation of the anaphase promoting complex/cyclosome (APC/C) and subsequent anaphase onset. SAC satisfaction is reversible, however, as addition of taxol during metaphase stops cyclin B1 degradation by the APC/C. We now show that targeting MAD1 to kinetochores during metaphase is sufficient to reestablish SAC activity after initial silencing. Using rapamycin-induced heterodimerization of FKBP-MAD1 to FRB-MIS12 and live monitoring of cyclin B1 degradation, we show that timed relocalization of MAD1 during metaphase can stop cyclin B1 degradation without affecting chromosome-spindle attachments. APC/C inhibition represented true SAC reactivation, as FKBP-MAD1 required an intact MAD2-interaction motif and MPS1 activity to accomplish this. Our data show that MAD1 kinetochore localization dictates SAC activity and imply that SAC regulatory mechanisms downstream of MAD1 remain functional in metaphase.Electronic supplementary materialThe online version of this article (doi:10.1007/s00412-014-0458-9) contains supplementary material, which is available to authorized users.

+Tip-Kinesin Complexes Steer Microtubule Growth In Vitro

Proper microtubule orientation in neurons is essential for targeting intracellular cargo to their correct destinations. However, the mechanisms by which uniform neuronal-microtubule organization is established and maintained are poorly understood. In vivo imaging in Drosophila dendrites showed that microtubules growing into branch points were consistently directed towards the cell body, thus maintaining their minus-end out orientation. RNAi knockdown experiments established that proper microtubule steering required EB1, APC and kinesin-2. Further two-hybrid assays indicated that APC interacts with both EB1 and the kinesin-2 subunit KAP3, forming a +TIP-kinesin complex. These findings led to the hypothesis that kinesin, concentrated at the plus-tips of growing microtubules by EB1, bends the growing microtubule by walking along static microtubules present at junctions. However, this mechanical role of EB1 is not consistent with the rapid kinetics reported for EB1 dissociating from growing microtubule plus-ends. To elucidate the minimum requirements for microtubule steering and investigate the mechanical activities of EB1 and kinesin during steering, we observed encounters between dynamic growing microtubule plus-ends and the lattice of stable microtubules on glass surfaces. To reconstitute +TIP-kinesin complexes in vitro, purified EB1 and kinesin were linked together through the Rapamycin-induced heterodimerization of FKBP-FRB. From gel filtration, the binding ratio of EB1 to kinesin ranged from 1.5:1 to 2:1. Under TIRF microscopy, EB1-kinesin complexes moved along microtubules and preferentially accumulated at growing microtubule plus-ends, indicating that association with EB1 promotes the localization of kinesin to growing plus-ends. More importantly, when +TIP-kinesin complex was present, the growing microtubules were bent and directed toward the plus-ends of immobilized microtubules, demonstrating that the +TIP-kinesin complex is sufficient to steer microtubule growth. We conclude that EB1 can localize kinesin to growing microtubule plus-ends and EB1 binding to microtubule plus-ends is sufficiently strong to steer microtubule growth.

Acute depletion of plasma membrane Phosphatidylinositol 4,5-bisphosphate impairs specific steps in G protein-coupled receptor endocytosis

Receptor endocytosis plays an important role in regulating the responsiveness of cells to specific ligands. Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] has been shown to be crucial for endocytosis of some cell surface receptors, such as EGF and transferrin receptors, but its role in G-protein-coupled receptor internalization has not been investigated. By using luciferase-labeled type 1 angiotensin II (AT1R), type 2C serotonin (5HT2CR) or β(2) adrenergic (β2AR) receptors and fluorescently tagged proteins (β-arrestin-2, plasma-membrane-targeted Venus, Rab5) we were able to follow the sequence of molecular interactions along the endocytic route of the receptors in HEK293 cells using the highly sensitive method of bioluminescence resonance energy transfer and confocal microscopy. To study the role of plasma membrane PtdIns(4,5)P(2) in receptor endocytosis, we used our previously developed rapamycin-inducible heterodimerization system, in which the recruitment of a 5-phosphatase domain to the plasma membrane degrades PtdIns(4,5)P(2). Here we show that ligand-induced interaction of AT1, 5HT2C and β(2)A receptors with β-arrestin-2 was unaffected by PtdIns(4,5)P(2) depletion. However, trafficking of the receptors to Rab5-positive early endosomes was completely abolished in the absence of PtdIns(4,5)P(2). Remarkably, removal of the receptors from the plasma membrane was reduced but not eliminated after PtdIns(4,5)P(2) depletion. Under these conditions, stimulated AT1 receptors clustered along the plasma membrane, but did not enter the cells. Our data suggest that in the absence of PtdIns(4,5)P(2), these receptors move into clathrin-coated membrane structures, but these are not cleaved efficiently and hence cannot reach the early endosomal compartment.

Rapid Inactivation of Proteins by Rapamycin-Induced Rerouting to Mitochondria

SummaryWe have developed a method for rapidly inactivating proteins with rapamycin-induced heterodimerization. Cells were stably transfected with siRNA-resistant, FKBP-tagged subunits of the adaptor protein (AP) complexes of clathrin-coated vesicles (CCVs), together with an FKBP and rapamycin-binding domain-containing construct with a mitochondrial targeting signal. Knocking down the endogenous subunit with siRNA, and then adding rapamycin, caused the APs to be rerouted to mitochondria within seconds. Rerouting AP-2 to mitochondria effectively abolished clathrin-mediated endocytosis of transferrin. In cells with rerouted AP-1, endocytosed cation-independent mannose 6-phosphate receptor (CIMPR) accumulated in a peripheral compartment, and isolated CCVs had reduced levels of CIMPR, but normal levels of the lysosomal hydrolase DNase II. Both observations support a role for AP-1 in retrograde trafficking. This type of approach, which we call a “knocksideways,” should be widely applicable as a means of inactivating proteins with a time scale of seconds or minutes rather than days.

Modulation of Cell Motility by Spatial Repositioning of Enzymatic ATP/ADP Exchange Capacity

ATP is the "principal energy currency" in metabolism and the most versatile small molecular regulator of cellular activities. Although already much is known about the role of ATP in fundamental processes of living systems, data about its compartmentalization are rather scarce, and we still have only very limited understanding of whether patterns in the distribution of intracellular ATP concentration ("ATP inhomogeneity") do exist and have a regulatory role. Here we report on the analysis of coupling of local ATP supply to regulation of actomyosin behavior, a widespread and dynamic process with conspicuous high ATP dependence, which is central to cell shape changes and cell motility. As an experimental model, we use embryonic fibroblasts from knock-out mice without major ATP-ADP exchange enzymes, in which we (re)introduce the ATP/ADP exchange enzyme adenylate kinase-1 (AK1) and deliberately manipulate its spatial positioning by coupling to different artificial location tags. By transfection-complementation of AK1 variants and comparison with yellow fluorescent protein controls, we found that motility and spreading were enhanced in cells with AK1 with a focal contact guidance tag. Intermediary enhancement was observed in cells with membrane-targeted or cytosolic AK1. Use of a heterodimer-inducing approach for transient translocation of AK1 to focal contacts under conditions of constant global AK1 activity in the cell corroborated these results. Based on our findings with these model systems, we propose that local ATP supply in the cell periphery and "on site" fuelling of the actomyosin machinery, when maintained via enzymes involved in phosphoryl transfer, are codetermining factors in the control of cell motility.

Regulation of PLCβ1a membrane anchoring by its substrate phosphatidylinositol (4,5)-bisphosphate

Basic knowledge as to the subcellular location and dynamics of PLCbeta isozymes is scant. Here, we report on the subcellular location of GFP-PLCbeta1a and the use of total internal reflection fluorescence (TIRF) microscopy to examine the dynamics of GFP-PLCbeta1a at the plasma membrane upon stimulation of Gq-coupled receptors. Using this technique, we observed PLCbeta1a dissociation from the plasma membrane upon addition of agonist. An increase in intracellular calcium and a decrease in PtdIns(4,5)P2 both coincided with a translocation of PLCbeta1a from the plasma membrane into the cytosol. In order to differentiate between calcium and PtdIns(4,5)P2, rapamycin-induced heterodimerization of FRB and FKBP12 fused to 5-phosphatase IV was used to instantaneously convert PtdIns(4,5)P2 into PtdIns(4)P. Addition of rapamycin caused PLCbeta1a to dissociate from the plasma membrane, indicating that removal of PtdIns(4,5)P2 is sufficient to cause translocation of PLCbeta1a from the plasma membrane. In conclusion, PLCbeta1a localization is regulated by its own substrate.

Modulation of translation-initiation in CHO-K1 cells by rapamycin-induced heterodimerization of engineered eIF4G fusion proteins.

Translation-initiation is a predominant checkpoint in mammalian cells which controls protein synthesis and fine-tunes the flow of information from gene to protein. In eukaryotes, translation-initiation is typically initiated at a 7-methyl-guanylic acid cap posttranscriptionally linked to the 5' end of mRNAs. Alternative cap-independent translation-initiation involves 5' untranslated regions (UTR) known as internal ribosome entry sites, which adopt a particular secondary structure. Translation-initiating ribosome assembly at cap or IRES elements is mediated by a multiprotein complex of which the initiation factor 4F (eIF4F) consisting of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G is a major constituent. eIF4G is a key target of picornaviral protease 2A, which cleaves this initiation factor into eIF4G(Delta) and (Delta)eIF4G to redirect the cellular translation machinery exclusively to its own IRES-containing transcripts. We have designed a novel translation control system (TCS) for conditional as well as adjustable translation of cap- and IRES-dependent transgene mRNAs in mammalian cells. eIF4G(Delta) and (Delta)eIF4G were fused C- and N-terminally to the FK506-binding protein (FKBP) and the FKBP-rapamycin-binding domain (FRB) of the human FKBP-rapamycin-associated protein (FRAP), respectively. Rapamycin-induced heterodimerization of eIF4G(Delta)-FKBP and FRB-(Delta)eIF4G fusion proteins reconstituted a functional chimeric elongation factor 4G in a dose-dependent manner. Rigorous quantitative expression analysis of cap- and IRES-dependent SEAP- (human placental secreted alkaline phosphatase) and luc- (Photinus pyralis luciferase) encoding reporter constructs confirmed adjustable translation control and revealed increased production of desired proteins in response to dimerization-induced heterologous eIF4G in Chinese hamster ovary (CHO-K1) cells.

Controlled dimerization of ErbB receptors provides evidence for differential signaling by homo- and heterodimers.

The four members of the ErbB family of receptor tyrosine kinases are involved in a complex array of combinatorial interactions involving homo- and heterodimers. Since most cell types express more than one member of the ErbB family, it is difficult to distinguish the biological activities of different homo- and heterodimers. Here we describe a method for inducing homo- or heterodimerization of ErbB receptors by using synthetic ligands without interference from the endogenous receptors. ErbB receptor chimeras containing synthetic ligand binding domains (FK506-binding protein [FKBP] or FKBP-rapamycin-binding domain [FRB]) were homodimerized with the bivalent FKBP ligand AP1510 and heterodimerized with the bifunctional FKBP-FRB ligand rapamycin. AP1510 treatment induced tyrosine phosphorylation of ErbB1 and ErbB2 homodimers and recruitment of Src homology 2 domain-containing proteins (Shc and Grb2). In addition, ErbB1 and ErbB2 homodimers activated downstream signaling pathways leading to Erk2 and Akt phosphorylation. However, only ErbB1 homodimers were internalized upon AP1510 stimulation, and only ErbB1 homodimers were able to associate with and induce phosphorylation of c-Cbl. Cells expressing AP1510-induced ErbB1 homodimers were able to associate with and induce phosphorylation of c-Cbl. Cells expressing AP1510-induced ErbB1 homodimers were able to form foci; however, cells expressing ErbB2 homodimers displayed a five- to sevenfold higher focus-forming ability. Using rapamycin-inducible heterodimerization we show that c-Cbl is unable to associate with ErbB1 in a ErbB1-ErbB2 heterodimer most likely because ErbB2 is unable to phosphorylate the c-Cbl binding site on ErbB1. Thus, we demonstrate that ErbB1 and ErbB2 homodimers differ in their abilities to transform fibroblasts and provide evidence for differential signaling by ErbB homodimers and heterodimers. These observations also validate the use of synthetic ligands to study the signaling and biological specificity of selected ErbB dimers in any cell type.