The mammalian endocytic cytoskeleton

Clathrin-mediated endocytosis (CME) is the major route through which cells internalise various substances and recycle membrane components. Via the coordinated action of many proteins, the membrane bends and invaginates to form a vesicle that buds off—along with its contents—into the cell. The contribution of the actin cytoskeleton to this highly dynamic process in mammalian cells is not well understood. Unlike in yeast, where there is a strict requirement for actin in CME, the significance of the actin cytoskeleton to mammalian CME is variable. However, a growing number of studies have established the actin cytoskeleton as a core component of mammalian CME, and our understanding of its contribution has been increasing at a rapid pace. In this review, we summarise the state-of-the-art regarding our understanding of the endocytic cytoskeleton, its physiological significance, and the questions that remain to be answered.     

Rice SCAMP1 defines clathrin-coated, trans-golgi-located tubular-vesicular structures as an early endosome in tobacco BY-2 cells

We recently identified multivesicular bodies (MVBs) as prevacuolar compartments (PVCs) in the secretory and endocytic pathways to the lytic vacuole in tobacco (Nicotiana tabacum) BY-2 cells. Secretory carrier membrane proteins (SCAMPs) are post-Golgi, integral membrane proteins mediating endocytosis in animal cells. To define the endocytic pathway in plants, we cloned the rice (Oryza sativa) homolog of animal SCAMP1 and generated transgenic tobacco BY-2 cells expressing yellow fluorescent protein (YFP)-SCAMP1 or SCAMP1-YFP fusions. Confocal immunofluorescence and immunogold electron microscopy studies demonstrated that YFP-SCAMP1 fusions and native SCAMP1 localize to the plasma membrane and mobile structures in the cytoplasm of transgenic BY-2 cells. Drug treatments and confocal immunofluorescence studies demonstrated that the punctate cytosolic organelles labeled by YFP-SCAMP1 or SCAMP1 were distinct from the Golgi apparatus and PVCs. SCAMP1-labeled organelles may represent an early endosome because the internalized endocytic markers FM4-64 and AM4-64 reached these organelles before PVCs. In addition, wortmannin caused the redistribution of SCAMP1 from the early endosomes to PVCs, probably as a result of fusions between the two compartments. Immunogold electron microscopy with high-pressure frozen/freeze-substituted samples identified the SCAMP1-positive organelles as tubular-vesicular structures at the trans-Golgi with clathrin coats. These early endosomal compartments resemble the previously described partially coated reticulum and trans-Golgi network in plant cells.     

A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis

Dual colour total internal reflection fluorescence microscopy is a powerful tool for decoding the molecular dynamics of clathrin-mediated endocytosis (CME). Typically, the recruitment of a fluorescent protein-tagged endocytic protein was referenced to the disappearance of spot-like clathrin-coated structure (CCS), but the precision of spot-like CCS disappearance as a marker for canonical CME remained unknown. Here we have used an imaging assay based on total internal reflection fluorescence microscopy to detect scission events with a resolution of ～ 2 s. We found that scission events engulfed comparable amounts of transferrin receptor cargo at CCSs of different sizes and CCS did not always disappear following scission. We measured the recruitment dynamics of 34 types of endocytic protein to scission events: Abp1, ACK1, amphiphysin1, APPL1, Arp3, BIN1, CALM, CIP4, clathrin light chain (Clc), cofilin, coronin1B, cortactin, dynamin1/2, endophilin2, Eps15, Eps8, epsin2, FBP17, FCHo1/2, GAK, Hip1R, lifeAct, mu2 subunit of the AP2 complex, myosin1E, myosin6, NECAP, N-WASP, OCRL1, Rab5, SNX9, synaptojanin2β1, and syndapin2. For each protein we aligned ～ 1,000 recruitment profiles to their respective scission events and constructed characteristic "recruitment signatures" that were grouped, as for yeast, to reveal the modular organization of mammalian CME. A detailed analysis revealed the unanticipated recruitment dynamics of SNX9, FBP17, and CIP4 and showed that the same set of proteins was recruited, in the same order, to scission events at CCSs of different sizes and lifetimes. Collectively these data reveal the fine-grained temporal structure of CME and suggest a simplified canonical model of mammalian CME in which the same core mechanism of CME, involving actin, operates at CCSs of diverse sizes and lifetimes.     

An Abp1-Dependent Route of Endocytosis Functions when the Classical Endocytic Pathway in Yeast Is Inhibited

Clathrin-mediated endocytosis (CME) is a well characterized pathway in both yeast and mammalian cells. An increasing number of alternative endocytic pathways have now been described in mammalian cells that can be both clathrin, actin, and Arf6- dependent or independent. In yeast, a single clathrin-mediated pathway has been characterized in detail. However, disruption of this pathway in many mutant strains indicates that other uptake pathways might exist, at least for bulk lipid and fluid internalization. Using a combination of genetics and live cell imaging, here we show evidence for a novel endocytic pathway in S. cerevisiae that does not involve several of the proteins previously shown to be associated with the ‘classic’ pathway of endocytosis. This alternative pathway functions in the presence of low levels of the actin-disrupting drug latrunculin-A which inhibits movement of the proteins Sla1, Sla2, and Sac6, and is independent of dynamin function. We reveal that in the absence of the ‘classic’ pathway, the actin binding protein Abp1 is now essential for bulk endocytosis. This novel pathway appears to be distinct from another described alternative endocytic route in S. cerevisiae as it involves at least some proteins known to be associated with cortical actin patches rather than being mediated at formin-dependent endocytic sites. These data indicate that cells have the capacity to use overlapping sets of components to facilitate endocytosis under a range of conditions.     

Crosstalk between Akt/GSK3β signaling and dynamin-1 regulates clathrin-mediated endocytosis

Clathrin-mediated endocytosis (CME) regulates signaling from the plasma membrane. Analysis of clathrin-coated pit (CCP) dynamics led us to propose the existence of a rate-limiting, regulatory step(s) that monitor the fidelity of early stages in CCP maturation. Here we show that nascent endocytic vesicles formed in mutant cells displaying rapid, dysregulated CME are defective in early endosomal trafficking, maturation and acidification, confirming the importance of this “checkpoint.” Dysregulated CME also alters EGF receptor signaling and leads to constitutive activation of the protein kinase Akt. Dynamin-1, which was thought to be neuron specific, is activated by the Akt/GSK3β signaling cascade in non-neuronal cells to trigger rapid, dysregulated CME. Acute activation of dynamin-1 in RPE cells by inhibition of GSK3β accelerates CME, alters CCP dynamics and, unexpectedly, increases the rate of CCP initiation. CRISPR-Cas9n-mediated knockout and reconstitution studies establish that dynamin-1 is activated by Akt/GSK3β signaling in H1299 non-small lung cancer cells. These findings provide direct evidence for an isoform-specific role for dynamin in regulating CME and reveal a feed-forward pathway that could link signaling from cell surface receptors to the regulation of CME.     

Yeast epsins contain an essential N-terminal ENTH domain, bind clathrin and are required for endocytosis.

The mammalian protein epsin is required for endocytosis. In this study, we have characterized two homologous yeast proteins, Ent1p and Ent2p, which are similar to mammalian epsin. An essential function for the highly conserved N-terminal epsin N-terminal homology (ENTH) domain was revealed using deletions and randomly generated temperature-sensitive ent1 alleles. Changes in conserved ENTH domain residues in ent1(ts) cells revealed defects in endocytosis and actin cytoskeleton structure. The Ent1 protein was localized to peripheral and internal punctate structures, and biochemical fractionation studies found the protein associated with a large, Triton X-100-insoluble pellet. Finally, an Ent1p clathrin-binding domain was mapped to the final eight amino acids (RGYTLIDL*) in the Ent1 protein sequence. Based on these and other data, we propose that the yeast epsin-like proteins are essential components of an endocytic complex that may act at multiple stages in the endocytic pathway.     

Receptor-mediated endocytosis in the Caenorhabditis elegans oocyte

The Caenorhabditis elegans oocyte is a highly amenable system for forward and reverse genetic analysis of receptor-mediated endocytosis. We describe the use of transgenic strains expressing a vitellogenin::green fluorescent protein (YP170::GFP) fusion to monitor yolk endocytosis by the C. elegans oocyte in vivo. This YP170::GFP reporter was used to assay the functions of C. elegans predicted proteins homologous to vertebrate endocytosis factors using RNA-mediated interference. We show that the basic components and pathways of endocytic trafficking are conserved between C. elegans and vertebrates, and that this system can be used to test the endocytic functions of any new gene. We also used the YP170::GFP assay to identify rme (receptor-mediated endocytosis) mutants. We describe a new member of the low-density lipoprotein receptor superfamily, RME-2, identified in our screens for endocytosis defective mutants. We show that RME-2 is the C. elegans yolk receptor.     

Control of lipid organization and actin assembly during clathrin-mediated endocytosis by the cytoplasmic tail of the rhomboid protein Rbd2

Clathrin-mediated endocytosis (CME) is facilitated by a precisely regulated burst of actin assembly. PtdIns(4,5)P2 is an important signaling lipid with conserved roles in CME and actin assembly regulation. Rhomboid family multipass transmembrane proteins regulate diverse cellular processes; however, rhomboid-mediated CME regulation has not been described. We report that yeast lacking the rhomboid protein Rbd2 exhibit accelerated endocytic-site dynamics and premature actin assembly during CME through a PtdIns(4,5)P2-dependent mechanism. Combined genetic and biochemical studies showed that the cytoplasmic tail of Rbd2 binds directly to PtdIns(4,5)P2 and is sufficient for Rbd2''s role in actin regulation. Analysis of an Rbd2 mutant with diminished PtdIns(4,5)P2-binding capacity indicates that this interaction is necessary for the temporal regulation of actin assembly during CME. The cytoplasmic tail of Rbd2 appears to modulate PtdIns(4,5)P2 distribution on the cell cortex. The syndapin-like F-BAR protein Bzz1 functions in a pathway with Rbd2 to control the timing of type 1 myosin recruitment and actin polymerization onset during CME. This work reveals that the previously unstudied rhomboid protein Rbd2 functions in vivo at the nexus of three highly conserved processes: lipid regulation, endocytic regulation, and cytoskeletal function.     

Regulation of cargo‐selective endocytosis by dynamin 2 GTPase‐activating protein girdin

In clathrin‐mediated endocytosis (CME), specificity and selectivity for cargoes are thought to be tightly regulated by cargo‐specific adaptors for distinct cellular functions. Here, we show that the actin‐binding protein girdin is a regulator of cargo‐selective CME. Girdin interacts with dynamin 2, a GTPase that excises endocytic vesicles from the plasma membrane, and functions as its GTPase‐activating protein. Interestingly, girdin depletion leads to the defect in clathrin‐coated pit formation in the center of cells. Also, we find that girdin differentially interacts with some cargoes, which competitively prevents girdin from interacting with dynamin 2 and confers the cargo selectivity for CME. Therefore, girdin regulates transferrin and E‐cadherin endocytosis in the center of cells and their subsequent polarized intracellular localization, but has no effect on integrin and epidermal growth factor receptor endocytosis that occurs at the cell periphery. Our results reveal that girdin regulates selective CME via a mechanism involving dynamin 2, but not by operating as a cargo‐specific adaptor.     

Fluorescence photobleaching analysis for the study of cellular dynamics

The wide availability of the confocal microscope and the emergence of green fluorescent protein (GFP) transfection technology has led to the increasing use of photobleaching studies to examine aspects of cellular dynamics in living cells. In this review, we examine the theory and practice of performing photobleaching studies using a confocal microscope. We illustrate the application of photobleaching protocols using our own measurements of fluorescently labelled red blood cells and of malaria parasite-infected erythrocytes expressing GFP fusions and examine other examples from the literature.     

Dynamics of Arabidopsis dynamin-related protein 1C and a clathrin light chain at the plasma membrane

Plant morphogenesis depends on polarized exocytic and endocytic membrane trafficking. Members of the Arabidopsis thaliana dynamin-related protein 1 (DRP1) subfamily are required for polarized cell expansion and cytokinesis. Using a combination of live-cell imaging techniques, we show that a functional DRP1C green fluorescent fusion protein (DRP1C-GFP) was localized at the division plane in dividing cells and to the plasma membrane in expanding interphase cells. In both tip growing root hairs and diffuse-polar expanding epidermal cells, DRP1C-GFP organized into dynamic foci at the cell cortex, which colocalized with a clathrin light chain fluorescent fusion protein (CLC-FFP), suggesting that DRP1C may participate in clathrin-mediated membrane dynamics. DRP1C-GFP and CLC-GFP foci dynamics are dependent on cytoskeleton organization, cytoplasmic streaming, and functional clathrin-mediated endocytic traffic. Our studies provide insight into DRP1 and clathrin dynamics in the plant cell cortex and indicate that the clathrin endocytic machinery in plants has both similarities and striking differences to that in mammalian cells and yeast.     

A Novel Dynamin-like Protein Associates with Cytoplasmic Vesicles and Tubules of the Endoplasmic Reticulum in Mammalian Cells

Abstract. Dynamins are 100-kilodalton guanosine triphosphatases that participate in the formation of nascent vesicles during endocytosis. Here, we have tested if novel dynamin-like proteins are expressed in mammalian cells to support vesicle trafficking processes at cytoplasmic sites distinct from the plasma membrane. Immunological and molecular biological methods were used to isolate a cDNA clone encoding an 80-kilodalton novel dynamin-like protein, DLP1, that shares up to 42% homology with other dynamin-related proteins. DLP1 is expressed in all tissues examined and contains two alternatively spliced regions that are differentially expressed in a tissue-specific manner. DLP1 is enriched in subcellular membrane fractions of cytoplasmic vesicles and endoplasmic reticulum. Morphological studies of DLP1 in cultured cells using either a specific antibody or an expressed green fluorescent protein (GFP)- DLP1 fusion protein revealed that DLP1 associates with punctate cytoplasmic vesicles that do not colocalize with conventional dynamin, clathrin, or endocytic ligands. Remarkably, DLP1-positive structures coalign with microtubules and, most strikingly, with endoplasmic reticulum tubules as verified by double labeling with antibodies to calnexin and Rab1 as well as by immunoelectron microscopy. These observations provide the first evidence that a novel dynamin-like protein is expressed in mammalian cells where it associates with a secretory, rather than endocytic membrane compartment.     

Selective caveolin-1-dependent endocytosis of glycosphingolipids

We studied the endocytosis of fluorescent glycosphingolipid (GSL) analogs in various cell types using pathway-specific inhibitors and colocalization studies with endocytic markers and DsRed caveolin-1 (cav-1). Based on inhibitor studies, all GSLs tested were internalized predominantly (>80%) by a clathrin-independent, caveolar-related mechanism, regardless of cell type. In addition, fluorescent lactosylceramide (LacCer) colocalized with DsRed-cav-1 in vesicular structures upon endocytosis in rat fibroblasts. The internalization mechanism for GSLs was unaffected by varying the carbohydrate headgroup or sphingosine backbone chain length; however, a fluorescent phosphatidylcholine analog was not internalized via caveolae, suggesting that the GSL ceramide core may be important for caveolar uptake. Internalization of fluorescent LacCer was reduced 80-90% in cell types with low cav-1, but was dramatically stimulated by cav-1 overexpression. However, even in cells with low levels of cav-1, residual LacCer internalization was clathrin independent. In contrast, cholera toxin B subunit (CtxB), which binds endogenous GM1, was internalized via clathrin-independent endocytosis in cells with high cav-1 expression, whereas significant clathrin-dependent uptake occurred in cells with low cav-1. Fluorescent GM1, normally internalized by clathrin-independent endocytosis in HeLa cells with low cav-1, was induced to partially internalize via the clathrin pathway in the presence of CtxB. These results suggest that GSL analogs are selectively internalized via a caveolar-related mechanism in most cell types, whereas CtxB may undergo "pathway switching" when cav-1 levels are low.     

ESCRT‐dependent protein sorting is required for the viability of yeast clathrin‐mediated endocytosis mutants

Endocytosis regulates many processes, including signaling pathways, nutrient uptake, and protein turnover. During clathrin‐mediated endocytosis (CME), adaptors bind to cytoplasmic regions of transmembrane cargo proteins, and many endocytic adaptors are also directly involved in the recruitment of clathrin. This clathrin‐associated sorting protein family includes the yeast epsins, Ent1/2, and AP180/PICALM homologs, Yap1801/2. Mutant strains lacking these four adaptors, but expressing an epsin N‐terminal homology (ENTH) domain necessary for viability (4Δ+ENTH), exhibit endocytic defects, such as cargo accumulation at the plasma membrane (PM). This CME‐deficient strain provides a sensitized background ideal for revealing cellular components that interact with clathrin adaptors. We performed a mutagenic screen to identify alleles that are lethal in 4Δ+ENTH cells using a colony‐sectoring reporter assay. After isolating candidate synthetic lethal genes by complementation, we confirmed that mutations in VPS4 led to inviability of a 4Δ+ENTH strain. Vps4 mediates the final step of endosomal sorting complex required for transport (ESCRT)‐dependent trafficking, and we found that multiple ESCRTs are also essential in 4Δ+ENTH cells, including Snf7, Snf8 and Vps36. Deletion of VPS4 from an end3Δ strain, another CME mutant, similarly resulted in inviability, and upregulation of a clathrin‐independent endocytosis pathway rescued 4Δ+ENTH vps4Δ cells. Loss of Vps4 from an otherwise wild‐type background caused multiple cargoes to accumulate at the PM because of an increase in Rcy1‐dependent recycling of internalized protein to the cell surface. Additionally, vps4Δ rcy1Δ mutants exhibited deleterious growth phenotypes. Together, our findings reveal previously unappreciated effects of disrupted ESCRT‐dependent trafficking on endocytic recycling and the PM.     

Let's think again about using mammalian temperature‐sensitive mutants to investigate functional molecules—The perspectives from the studies on three mutants showing chromosome instability

This review evaluates the use of temperature‐sensitive (ts) mutants to investigate functional molecules in mammalian cells. A series of studies were performed in which mammalian cells expressing functional molecules were isolated from ts mutants using complementation by the introduction and expression of the responsible protein tagged with the green fluorescent protein. The results showed that chromosome instability and cell‐cycle arrest were caused by ts defects in the following three molecules: the largest subunit of RNA polymerase II, a protein involved in splicing, and ubiquitin‐activating enzyme. The cells expressing functional protein were then isolated by introducing the responsible gene tagged with the green fluorescent protein to complement the ts phenotype. These cells proved to be useful in analyzing the dynamics of RNA polymerase II in living cells. Analyses of the functional interaction between proteins involved in splicing were also useful in the investigation of ts mutants and their derivatives. In addition, these cells demonstrated the functional localization of ubiquitin‐activating enzyme in the nucleus. Mammalian ts mutants continue to show great potential to aid in understanding the functions of the essential molecules in cells. Therefore, it is highly important that studies on the identification and characterization of the genes responsible for the phenotype of a mutant are carried out.     

Isoform and splice-variant specific functions of dynamin-2 revealed by analysis of conditional knock-out cells

Dynamin (Dyn) is a multifunctional GTPase implicated in several cellular events, including endocytosis, intracellular trafficking, cell signaling, and cytokinesis. The mammalian genome encodes three isoforms, Dyn1, Dyn2, and Dyn3, and several splice variants of each, leading to the suggestion that distinct isoforms and/or distinct splice variants might mediate distinct cellular functions. We generated a conditional Dyn2 KO cell line and performed knockout and reconstitution experiments to explore the isoform- and splice variant specific cellular functions of ubiquitously expressed Dyn2. We find that Dyn2 is required for clathrin-mediated endocytosis (CME), p75 export from the Golgi, and PDGF-stimulated macropinocytosis and cytokinesis, but not for other endocytic pathways. Surprisingly, CME and p75 exocytosis were efficiently rescued by reintroduction of Dyn2, but not Dyn1, suggesting that these two isoforms function differentially in vesicular trafficking in nonneuronal cells. Both isoforms rescued macropinocytosis and cytokinesis, suggesting that dynamin function in these processes might be mechanistically distinct from its role in CME. Although all four Dyn2 splice variants could equally restore CME, Dyn2ba and -bb were more effective at restoring p75 exocytosis. This splice variant specificity correlated with their differential targeting to the Golgi. These studies reveal isoform and splice-variant specific functions for Dyn2.     

The EH and SH3 domain Ese proteins regulate endocytosis by linking to dynamin and Eps15

Clathrin-mediated endocytosis is a multistep process which requires interaction between a number of conserved proteins. We have cloned two mammalian genes which code for a number of endocytic adaptor proteins. Two of these proteins, termed Ese1 and Ese2, contain two N-terminal EH domains, a central coiled-coil domain and five C-terminal SH3 domains. Ese1 is constitutively associated with Eps15 proteins to form a complex with at least 14 protein-protein interaction surfaces. Yeast two-hybrid assays have revealed that Ese1 EH and SH3 domains bind epsin family proteins and dynamin, respectively. Overexpression of Ese1 is sufficient to block clathrin-mediated endocytosis in cultured cells, presumably through disruption of higher order protein complexes, which are assembled on the endogenous Ese1-Eps15 scaffold. The Ese1-Eps15 scaffold therefore links dynamin, epsin and other endocytic pathway components.     

Coupling actin dynamics to the endocytic process in Saccharomyces cerevisiae

Summary. Endocytosis is an essential eukaryotic process that, in many systems, has been reported to require a functional actin cytoskeleton. The process of endocytosis is critical for controlling the protein–lipid composition of the plasma membrane and uptake of nutrients as well as pathogens and also plays an important role in regulation of cell signalling. While several distinct pathways for endocytosis have been characterised, all of these require remodelling of the cell cortex. The importance of a dynamic actin cytoskeleton for facilitating endocytosis has been recognised for many years in budding yeast and is increasingly supported by studies in mammalian cells. Current evidence suggests that cortical patches are sites of endocytosis in Saccharomyces cerevisiae and that these sites are composed of sequentially forming protein complexes. Distinct stages in complex formation are characterised by the presence of different activators of F-actin polymerisation. Disassembly of the complexes is also essential for the endocytosis to proceed. Mutants lacking the kinases Ark1 and Prk1 accumulate actin and endocytic machinery in a single large clump in cells. Phosphorylation of endocytic proteins including Sla1p is proposed to cause their removal from the complex and allow later stages of the invagination process to occur. Dephosphorylation of endocytic components may then allow subsequent reincorporation into new sites of endocytic complex assembly. Correspondence and reprints: Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom.     

Endocytic Accessory Proteins Are Functionally Distinguished by Their Differential Effects on the Maturation of Clathrin-coated Pits

Diverse cargo molecules (i.e., receptors and ligand/receptor complexes) are taken into the cell by clathrin-mediated endocytosis (CME) utilizing a core machinery consisting of cargo-specific adaptors, clathrin and the GTPase dynamin. Numerous endocytic accessory proteins are also required, but their differential roles and functional hierarchy during CME are not yet understood. Here, we used a combination of quantitative live-cell imaging by total internal reflection fluorescence microscopy (TIR-FM), and decomposition of the lifetime distributions of clathrin-coated pits (CCPs) to measure independent aspects of CCP dynamics, including the turnover of abortive and productive CCP species and their relative contributions. Capitalizing on the sensitivity of this assay, we have examined the effects of specific siRNA-mediated depletion of endocytic accessory proteins on CME progression. Of the 12 endocytic accessory proteins examined, we observed seven qualitatively different phenotypes upon protein depletion. From this data we derive a temporal hierarchy of protein function during early steps of CME. Our results support the idea that a subset of accessory proteins, which mediate coat assembly, membrane curvature, and cargo selection, can provide input into an endocytic restriction point/checkpoint mechanism that monitors CCP maturation.     

Imaging clathrin dynamics in Drosophila melanogaster hemocytes reveals a role for actin in vesicle fission

Clathrin-mediated endocytosis (CME) is essential for maintaining many basic cellular processes. We monitored the dynamics of clathrin in live Drosophila melanogaster hemocytes overexpressing clathrin light chain fused to enhanced green fluorescent protein (EGFP) using evanescent wave microscopy. Membrane-associated clathrin-coated structures (CCS) constitutively appeared at the peripheral filopodial membrane, moved centripetally while growing in intensity, before being eventually endocytosed within a few tens of seconds. This directed CCS traffic was independent of microtubules but could be blocked by latrunculin A. Taking advantage of available mutants of Drosophila, we expressed clathrin-EGFP in wasp and shibire mutant backgrounds to study the role of actin and dynamin in CCS dynamics and CME in hemocytes. We show that actin plays an essential role in CME in these cells, and that actin and dynamin act at the same stage, but independent of each other. Drosophila melanogaster hemocytes proved to be a promising model system to uncover the molecular events during CME in combining live-cell imaging and genetic analysis.