Journal of Cell Science

The microtubule and actin cytoskeletons form dynamic, interconnected networks that are critical for eukaryotic cell function. These networks govern intracellular organization, cargo transport, cell migration, and tissue morphogenesis. Microtubules and actin filaments are regulated by diverse binding proteins that control many aspects of their function. However, identifying cytoskeletal-interacting proteins has been challenging due to the transient and weak nature of many interactions and the disruption of native architecture by conventional biochemical approaches. These limitations suggest that numerous physiologically relevant cytoskeletal regulators remain undiscovered. Identifying these factors requires novel and sensitive methodologies that can capture cytoskeletal interactions under native cellular conditions. Here, we present MT-ID and Act-ID, powerful proximity-labeling tools for identifying microtubule and actin-interacting proteins, respectively. MT-ID employs the microtubule-binding domain of MAP7 (EMTB) fused to TurboID, a highly active promiscuous biotin ligase. Act-ID utilizes the actin-binding domain of ITPKA (F-tractin) similarly fused to TurboID. We validate both approaches by successfully identifying numerous known cytoskeletal regulators and discovering potentially novel interacting proteins. Functional characterization reveals that LIMCH1 is a previously unrecognized microtubule-associated protein whose depletion increases microtubule density. Additionally, we identify FBXO30 as a novel actin-interacting protein, with its loss promoting increased focal adhesion formation. MT-ID and Act-ID will be useful not only to identify cytoskeletal interacting proteins but also to define changes to the cytoskeletal interactome when cells are exposed to changing physiological conditions.

Heterotrimeric kinesin 2 is the canonical motor protein for anterograde intraflagellar transport (IFT), driving movement of protein complexes towards the tip of cilia and flagella. Here, we show that all members of the Euglenozoa group lack genes for heterotrimeric kinesins and instead possess a variable number of genes for two homodimeric kinesins termed KIN2A and KIN2B. When expressed in vitro, both Trypanosoma brucei kinesins form homodimers and move processively along brain microtubules, KIN2A being faster than KIN2B. Studies in T. brucei and Leishmania mexicana show anterograde and retrograde IFT of both kinesins, with KIN2A travelling throughout the whole length of the flagellum, while KIN2B is concentrated at its base. In the proximal portion of the flagellum, most KIN2B molecules travel without IFT proteins, except for a few particles that are associated with IFT proteins and reach the tip. Surprisingly, the absence of KIN2A has mild effects on IFT and flagellum assembly, whereas KIN2B is essential for both. Investigation of trypanosome flagella deprived of KIN2B revealed that IFT proteins do not access these flagella but that KIN2A can still circulate. These results support a division-of-labour model where KIN2B is responsible for the import of IFT proteins while KIN2A is responsible for most of the anterograde transport.

The spindle midzone, an array of overlapping, antiparallel microtubules, contributes to chromosome segregation and cytokinesis. As cells exit mitosis, midzone microtubules reorganize to form the midbody, the location of cell abscission. The mechanisms governing microtubule dynamics during this transition remain incompletely understood. The microtubule depolymerase, Kif2a, has been shown to contribute to midzone microtubule length control (Uehara et al., 2013), but how the depolymerase is regulated is not understood. Since CAMSAPs govern minus-end microtubule dynamics, we examined their role in midzone microtubule behavior. CAMSAP2, the major CAMSAP in HeLa cells, localized to the minus-ends of midzone microtubules and cells depleted of CAMSAP2, showed similar phenotypes as cells depleted of Kif2a, including elongated and bent midzones and enlarged asters. Next, we localized Kif2a in CAMSAP2-depleted cells and vice versa. CAMSAP2 remained present and extended along elongated midzone microtubules in Kif2a-depleted cells. In contrast Kif2a localization was no longer present at microtubule minus-ends but retained at plus-ends in CAMSAP2-depleted cells. In long-term live-cell movies of CAMSAP2-depleted cells abscission at the midbody was not detected, although two daughter cells formed. Markers for abscission including ESCRT-III component CHMP2A and Spastin were mislocalized, and midzone overlap zones, marked by PRC1, were extended. Together, our results demonstrate that CAMSAP2 is essential for midzone microtubule organization and dynamics, ultimately impacting cell abscission.

Although calmodulin is best known as an intracellular calcium sensor, it also possesses calcium-independent functions in unicellular organisms. This is exemplified by the budding yeast S. cerevisiae calmodulin, which binds its essential targets, the pericentrin-like protein Spc110 and type I and V myosins, without needing calcium. Whether such calcium-independent cellular functions are conserved in other yeasts and vertebrates nevertheless remains an open question. Here, we examined the calcium-independent functions of the fission yeast S. pombe calmodulin Cam1 by measuring its intracellular distribution. Using quantitative fluorescence microscopy, we assessed the intracellular localization of two cam1 mutants, where binding of Ca2+ had been compromised by mutations in their EF hands, compared to the wild type protein. Both Cam1-2V and -3V reduced their localization by 90% to the yeast microtubule-organizing center spindle pole bodies (SPB). In contrast, these two mutants did not affect the myosin-dependent localization to the equatorial division plane and to the cell tips. Replacing the endogenous cam1 with cam1-2V decreased the SPB localization of pericentrin Pcp1 by 69%, without changing the localization of either type V or I myosins. Over-expression of Pcp1 rescued the mitotic defects of cam1-2V cells at the restrictive temperature. Surprisingly, the cytokinesis of this cam1 mutant was largely normal. We concluded that fission yeast calmodulin Cam1 depends on Ca2+to be a component of SPBs, suggesting that calcium plays a critical role in the assembly of SPBs.

Cadmium, being a highly toxic metal, perturbs cellular homeostasis by forming stable complexes with numerous thiol-active proteins, ultimately leading to severe liver and lung damage. Despite its well-documented toxicity, the molecular mechanisms governing cadmium export remain poorly understood. Given the chemical similarity between cadmium and copper, we investigated whether the canonical copper-exporting ATPases, ATP7A and ATP7B participate in cadmium handling. Upon Cd treatment in hepatocytes, ATP7B undergoes trafficking to lysosomes via the retromer complex, as also observed in the case of elevated copper, accompanied by the upregulation of acidic lysosomal populations. In contrast, ATP7A expressed in lung adenocarcinoma cells, though exhibit vesicular redistribution upon Cd exposure, does not mediate lysosomal sequestration, suggesting distinct deployment of late secretory pathways by the two copper ATPases in response to cadmium. We have also observed that ATP7B-/- hepatocytes exhibit increased sensitivity to Cd exposure compared to wild-type cells. Whereas, overexpressing the ATP7B amino-terminal copper-binding domain in bacteria alleviates cadmium-induced stress, indicating its capacity to sequester Cd. Caenorhabditis elegans lacking copper-ATPase cua-1, displayed increased Cd sensitivity, while mutants (glo-1-/-), deficient in lysosome-related organelles (LRO), and (lmp-1-/-), deficient in lysosomal membrane glycoprotein, showed reduced resistance to cadmium toxicity. Treatment of the worm with cadmium increases the abundance of lysosomes marked by elevation in lysosomal biogenesis and functional genes, reinforcing the importance of lysosomal pathways in cadmium detoxification. To summarise, we delineated the non-canonical role of copper ATPases and lysosomes in cadmium-induced cellular toxicity.

Abnormalities in nuclear morphology are an important diagnostic tool to determine malignancy in cancer cells and are characterised by nuclear blebbing and deformations. Nuclear shape is mostly maintained by a dense protein meshwork of lamins, consisting of 4 lamin subtypes, of which the individual contribution to nuclear shape maintenance remains elusive. In this study, we decouple the roles of lamin A, C, and B1 across cancer cell lines with varying malignant potential (HeLa, HT1080, and MDA-MB-231). Using single-cell correlation analysis, we directly link reduced lamin A/C, and not lamin B1, expression levels to nuclear deformability. We found that the nuclear shape of the more malignant MDA-MB-231 cells is approximately 4-fold more sensitive to lamin A/C than HeLa and HT1080 cells. Biochemical analyses reveal cell-type-specific variation in lamin A/C interactions and homodimer formation that correlates with nuclear shape deformations. In contrast to healthy mouse embryonic fibroblast cells, malignant cells exhibit reduced dimerisation, which correlates with nuclear deformability. As such, our study links, for the first time, the lamin A/C dimerisation state to nuclear abnormalities, thereby providing new avenues for investigating cancer progression.

Actin superfamily members are critical for the biology of eukaryotes and archaea. Actin-related proteins (Arps) are a subgroup within the actin superfamily and play essential roles in trafficking, replication and motility. The genome of the malaria parasite Plasmodium contains a set of Arps unique to apicomplexans, termed actin-like proteins (Alps). However, the importance and specific roles of many of these Alps in Plasmodium progression are not yet understood. Here, we determined the functional contribution of Plasmodium berghei Alp3 and Alp5a (recently relabelled as Arp3) by generation of knock-out (KO) lines and their subsequent characterisation across different life cycle stages. Deletion of either Alp did not affect blood stage growth, gametogenesis and ookinete gliding motility. However, deletion of Alp5a lead to smaller and fewer oocysts as well as severely impaired sporozoite formation. The Alp3KO line had highly reduced oocyst loads compared to wild-type parasites. This striking decrease was due to impaired ookinete penetration of the mosquito midgut epithelium. Our study shows that both Alp3 and Alp5a are indispensable for Plasmodium transmission at different steps of initial mosquito infection, provides insights into the role of specific unique members of the actin superfamily during parasite progression and the requirements for efficient midgut penetration.

Eukaryotic cells control their size by coordinating growth and division. Fission yeast divide at a reproducible cell size due to regulated activation of the cyclin-dependent kinase Cdk1. The nuclear concentration of mitotic cyclin Cdc13 increases in a time-dependent manner to promote Cdk1 activation as cells grow. Here, we show that interphase Cdc13 is stable against degradation and nuclear export, but is diluted by cell growth. Low glucose reduced cell growth rate but not time-dependent accumulation of Cdc13. Uncoupling the rates of cell growth and Cdc13 accumulation resulted in higher concentrations of nuclear Cdc13 despite reduced cell size. This change coincided with reduced activating phosphorylation of Cdk1-T167 and occurred dynamically during abrupt changes in glucose concentration. Mathematical modeling and experiments showed that cells maintain size homeostasis under these conditions. In contrast to low glucose, poor nitrogen reduced both cell growth rate and Cdc13 accumulation rate. Therefore, Cdc13 accumulation is independent of cell growth rate but can be altered by nutrient-specific mechanisms.

Cell migration depends on coordinating cell shape changes with force generation, yet how these processes are integrated remains unclear. Here, we combine live-cell imaging with traction force microscopy and computational analysis to quantify cell morphology, motility and force generation in migrating fibroblasts. We find that traction force magnitudes display a multimodal distribution, suggesting discrete migratory regimes. Using a Hidden Markov Model, we identify distinct force states that exhibit differences in shape and motion metrics, and show that individual cells transition between force states over time. To test the role of cytoskeletal organization in establishing the identified states, we analyzed cells lacking Arpc2, which disrupts branched actin assembly. Despite reduced forces and altered morphology, these cells also exhibit three migratory states. State transitions occur more frequently in cells lacking Arpc2 and unlike normal cells their protrusion geometry is force dependent. Together, our findings show that cell migration is organized into discrete mechanical states that couple morphology, motility and force generation. SUMMARY STATEMENTFibroblast motility involves distinct migratory states. These states exist independent of branched actin. However, state transition frequencies, traction force magnitudes and protrusion geometry are branched actin dependent.

BackgroundSkin is constantly exposed to mechanical forces such as pressure and friction, which need to be sensed and buffered to ensure tissue homeostasis and barrier function. Desmosomes are essential for epidermal integrity, but their role in converting mechanical cues into cellular signaling responses are not well understood. MethodsHere, we combine proteomics and shear-stress assays with live-cell reporters to investigate how desmosomes modulate stress-kinase pathways in keratinocytes. ResultsWe show that the desmosomal adhesion molecule DSG3 is essential not only for cell-cell adhesion but also for modulating p38MAPK and ERK signaling. Loss of DSG3 disrupts mechanotransduction-related protein networks, including the expression of the mechanosensitive channel Piezo1. Under static conditions, DSG3 dampens ERK activity via Piezo1-dependent mechanisms, whereas DSG3 suppresses p38MAPK activity through an independent mechanism. In contrast, DSG3 is required to trigger an activation of both ERK and p38MAPK in response to shear stress in a Piezo1-dependent manner. Experiments with domain-specific DSG3 mutants demonstrate that cell cohesion and signaling responses are partially uncoupled, while maintaining DSG3 tail integrity was crucial for p38MAPK and ERK responses. ConclusionThese findings demonstrate that DSG3 independently coordinates adhesion and mechanotransduction in a domain-specific manner, providing novel insights into how DSG3 contributes to epithelial integrity under dynamic mechanical environments.

To properly respond to their environment, cells adjust the activity of key regulatory proteins and rates of gene expression. Methods to detect and quantify these forms of regulatory dynamics in living cells are of central importance for understanding cellular signaling events in both physiological and pathological conditions. Current technologies in this field make use of fluorescent probes to track cell signaling dynamics. Although these technologies have been used for decades, challenges remain. In particular, the segmentation, tracking, and interpretation of single cell dynamic data are time-consuming, prone to subjective errors, and often lacking in standardization across experiments. Here, we present SPIFEE, a data pipeline that uses experiment-dependent parameters to smooth noise and quantify key features of fluorescence data from time-lapse imaging studies. Processing data in this manner enhances and accelerates quantification of live-cell gene and protein expression, simplifies data analysis, and facilitates hypothesis generation. Author SummaryCells adjust protein activity and gene expression levels over time to respond to changes in their environment, a process referred to as cell signaling dynamics. Quantifying cell signaling dynamics in living cells often uses fluorescent probes, such as green fluorescent protein (GFP) and its spectral variants, to track changes in gene expression or protein activity over time. Challenges inherent in analyzing fluorescence data from single cells stem from biological and experimental noise, time-consuming quantification, and subjective errors. To address these challenges, we developed a computational tool called Signal Processing and Integrated Feature Extraction (SPIFEE). The pipeline improves the quality of fluorescence data analysis by reducing noise and extracting signal features in a way that is both intuitive and objective. The pipeline provides more accurate, rapid, and unbiased quantification of time-lapse microscopy data.

Cell polarity, essential for cell development and function, relies on dynamic subcellular distribution of structural and signaling molecules. Tip growth, an extreme form of polar growth, involves unidirectional expansion at the apical region of cells and requires precise spatiotemporal coordination to achieve periodic and directional growth. Understanding their spatiotemporal dynamics is critical for elucidating mechanisms and functions of cell polarity. However, manual quantification of such dynamics is extremely time-consuming, hindering advancements in the field. Current algorithms have limited power and flexibility in analyzing the distribution and dynamics of molecules and structures, particularly for tip-growing cells with oscillatory and dynamic behavior. To address this challenge, we present TipQuant, an automated analysis tool that robustly detects tips and analyzes spatiotemporal dynamics of fluorescently labeled molecules/structures on plasma membranes and in cytoplasm at apices of tip-growing cells, enabling quantitative understanding of signaling and structural components in these systems.

In Caenorhabditis elegans embryos, the nuclear transport receptor IMB-2 (Importin Beta Family-2, a karyopherin {beta}2) preferentially localizes to the nuclear envelope along with its encoding mRNA. This suggests that imb-2 mRNA is locally translated at the nuclear envelope. To test whether imb-2s two putative human orthologs, Transportin 1 (TNPO1) and Transportin 2 (TNPO2), exhibited similar mRNA localization and local translation, we performed smiFISH and microscopy in U2OS, HeLa, and human pluripotent stem cells. Neither human TNPO1 nor TNPO2 mRNA localized to the nuclear envelope in any tested human cell type. However, the human TNPO1 protein and the C. elegans IMB-2 protein both localized to the nucleus and its periphery. This suggests that despite their shared functional roles and high amino acid sequence identities (52% and 51%, respectively), these karyopherins differed in their translational dynamics.

Telomere-led rapid prophase chromosome movements (RPMs) during meiotic prophase are critical for homologous chromosome pairing and proper meiotic progression. These movements are generated by the cytoskeleton and are transmitted to the telomeres via the LINC complex, yet the cytoplasmic components that generate these forces remain poorly defined. Among candidates of microtubule-associated motor proteins in mouse primary spermatocytes, we confirmed KIF5B as a specific interactor of the KASH5-LINC complex. Total internal reflection fluorescence microscopy and microtubule sedimentation assays performed with purified recombinant proteins suggest a direct interaction between KASH5 and KIF5B on microtubules, enhanced by MAP7, a known KIF5B-recruiting and activating cofactor. Mapping the KIF5B-binding surface of KASH5 revealed that KASH5 N-terminal EF-hand domains mediate the interaction. Further, in vivo KIF5B-KASH5 interaction and KIF5B role in RPMs are evidenced as (1) KIF5B is recruited by KASH5-SUN1 to the nuclear envelope in two different cultured somatic cell models, (2) KIF5B is telomere-associated and colocalizes with KASH5, and microtubules associated with the nuclear envelope in mouse spermatocytes, and (3) chemical inhibition of KIF5B reduces telomere-led chromosome motions. Altogether, our findings identify the KIF5B kinesin as a previously unrecognized component of the force-generating machinery that drives chromosome movement during meiotic prophase I, acting through KASH5 as a specific nuclear membrane adaptor.

The Ezrin, Radixin, and Moesin (ERM) family of proteins anchors the actin cytoskeleton to the plasma membrane for the purpose of either stabilizing or altering cell shape. In Caenorhabditis elegans, ERM-1, is essential for cell polarity, signaling, intestine development, and larval viability. Interestingly, ERM-1 proteins are produced by erm-1 mRNA transcripts that concentrate at the plasma membrane in embryos. The localization of erm-1 mRNA to the plasma membrane occurs in a 3UTR-independent, translation-dependent manner, directed by the PH-subdomain within ERM-1s N-terminal FERM domain. This has led to the model that erm-1 mRNA, its associated ribosome, and its emerging nascent peptide are all transported together to the plasma membrane as a complex. Here, we characterize the transport mechanism. Using a microscopy approach, we observed that the localizations of erm-1 mRNA and ERM-1 protein to the plasma membrane were disrupted by nocodazole treatment, illustrating a microtubule role. Furthermore, erm-1 mRNA and ERM-1 protein localized to the plasma membrane independently of myosin and dynein motors, but dependent on the kinesin bmk-1 (bmk-1), a plus-end-directed, Kinesin-5 family motor protein. Loss of bmk-1 did not reduce the total number of erm-1 mRNA molecules in the cell, arguing against a diffusion- and protection-based mechanism of mRNA localization. Together, these findings suggest that erm-1 mRNA is localized via an active transport pathway mediated by a plus-end-directed kinesin adapter. Interestingly, loss of bmk-1 led to diffuse localization of ERM-1 protein along the plasma membrane and reduced ERM-1 protein levels at the site of abscission, the midbody, and the midbody remnant. This suggests that ERM-1 local translation at the plasma membrane is critical for its proteins ultimate spatial patterning in the cell.

Along with the membrane potential and respiration, mitochondrial matrix volume is a critical parameter that determines mitochondrial function. Mitochondria undergo constant changes in matrix volume and cristae dynamics, and in processes that are critical for normal metabolic rates and pathophysiological responses. Changes in matrix volume cannot be easily measured by conventional fluorescence imaging techniques due to the size of the sub-organellar structures, which are below resolution. This challenge was successfully resolved in studies of isolated mitochondria with the use of scattered light. Here we use dark-field imaging, which relies on scattered light contrast, to measure matrix volume dynamics in living cells. We demonstrate that mitochondrial volume changes can be easily detected as changes in intensity of the scattered light following matrix volume modulation with K+ ionophores or by onset of the permeability transition. Specifically, we found that stimulation of K+ influx leads to increase of mitochondrial matrix volume while stimulation of K+ efflux leads to matrix shrinkage, and that activation of the permeability transition leads to high-amplitude mitochondrial swelling in wild-type but not in cells lacking subunit c of ATP synthase. These results directly demonstrate the dynamic nature of mitochondrial matrix volume and its link to physiological and pathological ion transport.

Chromosome segregation fidelity during meiosis is critical for genome integrity, with aneuploidy causing infertility, miscarriages, and congenital anomalies. In the oocytes of many species, spindle assembly occurs in the absence of centrosomes that normally function as microtubule-organizing centers at the poles. Such acentrosomal spindles are believed to pose significant challenges for accurate chromosome segregation compared to centrosomal organized spindles. Previous work in Drosophila has shown that the chromosomal passenger complex (CPC) is required for acentrosomal spindle assembly. We found that heterochromatin protein-1 (HP1) plays a critical role in regulating CPC localization and spindle assembly. Furthermore, HP1 moves to the microtubules, where it has roles in building a functional spindle and interacts with the CPC to regulate chromosome biorientation. These results indicate that spindle assembly is mediated by multiple interactions between the CPC, HP1, and the chromosomes, and provide insights into the mechanisms that restricts spindle assembly to the chromosomes in Drosophila oocytes.

Cells migrate with varying degrees of polarization and directional persistence as exemplified by epithelial, mesenchymal and amoeboid cell types. Depending on the physiological and developmental context, these states are often interchangeable, reflecting the plastic and adaptive nature of the cytoskeleton. However, general principles governing such motility-mode transitions remain poorly established, and it is unclear whether they apply to non-metazoan cells. Here, we report previously overlooked features of the amoebozoan Dictyostelium discoideum, demonstrating that it undergoes pronounced adhesion-dependent changes in both motility and morphology. Unlike the well-known pseudopodia-rich forms observed on weakly adhesive surfaces, cells on highly adhesive substrates adopt fan-shaped morphologies reminiscent of cultured mesenchymal cells. These cells are characterized by lamellipodia-like protrusions enriched in the SCAR/WAVE complex, large focal adhesion-like plaques, F-actin-independent front-rear gradients of Ras/Rap activity. Furthermore, they exhibit a marked increase in cortical stiffness dependent on F-actin, talins, and the RhoA homolog RacE. Their high directional persistence depends on the persistent localization of the SCAR/WAVE complex, talin-mediated substrate anchoring, and RacE-dependent stabilization of the cell rear. We propose that adhesion-engaged remodeling of cell polarity and cortical mechanics is an evolutionarily ancient feature that predates the specialization of adhesion receptors.

Synaptic vesicle proteins (SVPs) are synthesised in the neuronal soma trafficked as precursor synaptic vesicles (pre-SVs) on route to synapses. While pre-SVs are known to have heterogeneous protein composition and can co-traffic with lysosomal proteins. In this study, we assess the trafficking routes and kinetics of Synatobrevin-1 (SNB-1) released from the ER using the RUSH system in vivo in C. elegans touch receptor neurons. We showed that ER-released SNB-1 follows at least two temporally distinct trafficking routes. A predominantly anterogradely moving population of SNB-1 carrying vesicles appeared early, within 20 minutes of ER release in the axon without overlap with lysosomal proteins. Another SNB-1 population at 45 minutes post-ER release overlapped with endolysosomal compartments in both the cell body and the axon. Early SNB-1 carrying vesicles co-migrate with a transmembrane synaptic vesicle protein Synaptogyrin (SNG-1) and RAB-27 but fewer with RAB-3, suggesting that SVPs can be co-sorted into the same carriers prior to overlap with lysosomal proteins. The SV-lysosomal protein overlap occurs even when SNB-1 endocytosis on the plasma membrane is reduced in unc-11/ap180 mutants. Finally, we identified SAM-4/Myrlysin, a subunit of the BORC complex, as a regulator of both the trafficking kinetics of Synaptobrevin-1 intermediates and the cargo composition of pre-SVs. Loss of SAM-4 accelerated SV-lysosomal protein overlap and reduced co-transport of SNG-1 with SNB-1 in early pre-SVs in the axon. Together, these findings reveal heterogeneity in pre-SV biogenesis routes and identify SAM-4 as a key regulator of both the kinetics and cargo composition of synaptic vesicle precursors.

During maturation of a Golgi cisterna, multiple vesicular transport pathways recycle resident Golgi proteins. Recycling vesicles are captured by Golgi-associated tethers. To assign individual tethers to specific recycling pathways in Saccharomyces cerevisiae, we examined tether arrival and departure using kinetic mapping, and we examined tether function using an ectopic tether localization assay. Those approaches yielded mutually consistent results. Our analysis focused on two coiled coil golgin tethers and the multi-subunit tether GARP. At an intermediate stage of cisternal maturation, the golgin Sgm1 tethers proteins that follow an intra-Golgi recycling pathway dependent on COPI. At a late stage of cisternal maturation, GARP and the golgin Imh1 tether trans- Golgi network (TGN) proteins that follow an intra-Golgi recycling pathway dependent on the AP-1 and Ent5 clathrin adaptors. This involvement of GARP in intra-Golgi recycling had not previously been documented. Imh1 also tethers proteins that recycle from prevacuolar endosome compartments to the TGN. Our findings contribute to an integrated model of Golgi membrane traffic.