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Signaling through the Ror2 receptor tyrosine kinase promotes invadopodia formation for tumor invasion. Here, we identify intraflagellar transport 20 (IFT20) as a new target of this signaling in tumors that lack primary cilia, and find that IFT20 mediates the ability of Ror2 signaling to induce the invasiveness of these tumors. We also find that IFT20 regulates the nucleation of Golgi-derived microtubules by affecting the GM130-AKAP450 complex, which promotes Golgi ribbon formation in achieving polarized secretion for cell migration and invasion. Furthermore, IFT20 promotes the efficiency of transport through the Golgi complex. These findings shed new insights into how Ror2 signaling promotes tumor invasiveness, and also advance the understanding of how Golgi structure and transport can be regulated.

Endocytic recycling, for some time considered to occur by default, is now emerging as an active sorting process. These studies are increasing our understanding of the physiological events that require recycling. Endocytic recycling returns proteins to the plasma membrane in many physiological contexts. Studies of these events have helped to elucidate fundamental mechanisms that underlie recycling. Recycling was for some time considered to be the exception to a general mechanism of active cargo sorting in multiple intracellular pathways. In recent years, studies have begun to reconcile this seeming disparity and also suggest explanations for why early recycling studies did not detect active sorting. Further articulation of this emerging trend has far-reaching implications for a deeper understanding of many physiological and pathological events that require recycling.

The epidermal growth factor receptor (EGFR) plays important roles in multiple cellular events, including growth, differentiation, and motility. A major mechanism of downregulating EGFR function involves its endocytic transport to the lysosome. Sorting of proteins into intracellular pathways involves cargo adaptors recognizing sorting signals on cargo proteins. A dileucine-based sorting signal has been identified previously for the sorting of endosomal EGFR to the lysosome, but a cargo adaptor that recognizes this signal remains unknown. Here, we find that phosphoglycerate kinase 1 (PGK1) is recruited to endosomal membrane upon its phosphorylation, where it binds to the dileucine sorting signal in EGFR to promote the lysosomal transport of this receptor. We also elucidate two mechanisms that act in concert to promote PGK1 recruitment to endosomal membrane, a lipid-based mechanism that involves phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and a protein-based mechanism that involves hepatocyte growth factor receptor substrate (Hrs). These findings reveal an unexpected function for a metabolic enzyme and advance the mechanistic understanding of how EGFR is transported to the lysosome. The epidermal growth factor receptor (EGFR) plays important roles in cell growth and motility. Here, authors reveal an unexpected function for a metabolic enzyme PGK1 and advance the mechanistic understanding of lysosomal transport of EGFR.

Coat proteins have a central role in vesicular transport by binding to cargoes for their sorting into intracellular pathways. Cargo recognition is mediated by components of the coat complex known as adaptor proteins1–3. We previously showed that Arf-GAP with coil-coil, ANK repeat and PH domain-containing protein 1 (ACAP1) functions as an adaptor for a clathrin coat complex that has a function in endocytic recycling4–6. Here, we show that the protein kinase Akt acts as a co-adaptor in this complex, and is needed in conjunction with ACAP1 to bind to cargo proteins to promote their recycling. In addition to advancing the understanding of endocytic recycling, we uncover a fundamentally different function in which a kinase acts, as Akt in this case is an effector rather than a regulator in a cellular event.Hsu et al. find that the protein kinase Akt acts as a co-adaptor in the clathrin coat complex and is needed in conjunction with ACAP1 to bind to cargo proteins to promote their recycling.

The aldehyde dehydrogenase (ALDH) family of metabolic enzymes converts aldehydes to carboxylates. Here, we find that the reductive consequence of ALDH7A1 activity, which generates NADH (nicotinamide adenine dinucleotide, reduced form) from NAD, underlies how ALDH7A1 coordinates a broad inhibition of the intracellular transport pathways. Studying vesicle formation by the Coat Protein I (COPI) complex, we elucidate that NADH generated by ALDH7A1 targets Brefeldin-A ADP-Ribosylated Substrate (BARS) to inhibit COPI vesicle fission. Moreover, defining a physiologic role for the broad transport inhibition exerted by ALDH7A1, we find that it acts to reduce energy consumption during hypoxia and starvation to promote cellular energy homeostasis. These findings advance the understanding of intracellular transport by revealing how the coordination of multiple pathways can be achieved, and also defining circumstances when such coordination is needed, as well as uncovering an unexpected way that NADH acts in cellular energetics. Intracellular vesicle transport can be regulated by Brefeldin‐A ADP‐Ribosylated Substrate (BARS) during vesicle fission. Here, the authors show that NADH generated by aldehyde dehydrogenase 7A1 (ALDH7A1) inhibits intracellular transport by targeting BARS and inhibiting COPI vesicle fission during situations of energy deprivation

The COPI complex, which has a role in retrograde transport through the Golgi, is shown to also mediate anterograde tubular transport through the Golgi; in response to external stimuli, the small GTPase CDC42 acts as an essential modulator of bidirectional Golgi transport, and promotes the sorting of cargoes destined for anterograde transport into the tubules at the expense of those targeted for retrograde transport. Two-way traffic through Golgi complex tubules The Golgi complex consists of a series of plate-like stacks, connected by structures called tubules. It has been called the cell's 'post office', given its central role in the sorting and distribution of proteins to their proper intracellular destination. Here Victor Hsu and colleagues provide evidence that Golgi tubules play a role in anterograde transport from Golgi towards plasma membrane — a process previously thought to be mediated primarily by the Golgi stacks. They find that the COPI protein complex, known to play a part in retrograde transport through the Golgi, also mediates anterograde tubular transport. This raises the question of how COPI can sort cargoes for transport in opposite directions. The authors find that in response to external stimuli, the small GTPase CDC42 is an essential modulator of bidirectional Golgi transport, promoting the sorting of cargoes destined for anterograde transport into the tubules at the expense of those targeted for retrograde transport. The Golgi complex has a central role in the intracellular sorting of secretory proteins 1 , 2 . Anterograde transport through the Golgi has been explained by the movement of Golgi cisternae, known as cisternal maturation 3 , 4 , 5 . Because this explanation is now appreciated to be incomplete 6 , interest has developed in understanding tubules that connect the Golgi cisternae 7 , 8 , 9 . Here we show that the coat protein I (COPI) complex sorts anterograde cargoes into these tubules in human cells. Moreover, the small GTPase CDC42 regulates bidirectional Golgi transport by targeting the dual functions of COPI in cargo sorting and carrier formation. CDC42 also directly imparts membrane curvature to promote COPI tubule formation. Our findings further reveal that COPI tubular transport complements cisternal maturation in explaining how anterograde Golgi transport is achieved, and that bidirectional COPI transport is modulated by environmental cues through CDC42.

Cellular levels of phosphatidylinositol 4-phosphate (PI4P) have been shown to be upregulated during RNA replication of several viruses, including the HCV replicon model. However, whether PI4P is required in an infectious HCV model remains unknown. Moreover, it is not established whether the host transport machinery is sequestered by the generation of PI4P during HCV infection. Here we found that PI4P was enriched in HCV replication complexes when Huh7.5.1 cells were infected with JFH1. HCV replication was inhibited upon overexpression of the PI4P phosphatase Sac1. The PI4P kinase PI4KIIIβ was also found to be required for HCV replication. Moreover, the vesicular transport proteins ARF1 and GBF1 colocalized with PI4KIIIβ and were both required for HCV replication. During authentic HCV infection, PI4P plays an integral role in virus replication.

Studies on vesicle formation by the Coat Protein I (COPI) complex have contributed to a basic understanding of how vesicular transport is initiated. Phosphatidic acid (PA) and diacylglycerol (DAG) have been found previously to be required for the fission stage of COPI vesicle formation. Here, we find that PA with varying lipid geometry can all promote early fission, but only PA with shortened acyl chains promotes late fission. Moreover, diacylglycerol (DAG) acts after PA in late fission, with this role of DAG also requiring shorter acyl chains. Further highlighting the importance of the short-chain lipid geometry for late fission, we find that shorter forms of PA and DAG promote the vesiculation ability of COPI fission factors. These findings advance a general understanding of how lipid geometry contributes to membrane deformation for vesicle fission, and also how proteins and lipids coordinate their actions in driving this process. Phosphatidic acid and diacylglycerol are required for COPI vesicle fission. Here, the authors find that shorter forms of these lipids promote the late stage of COPI vesicle fission, further suggesting how lipid geometry contributes to membrane deformation in driving fission.

Intracellular recycling pathways play critical roles in internalizing membrane and fluid phase cargo and in balancing the inflow and outflow of membrane and cell surface molecules. To identify proteins involved in the regulation of endocytic recycling, we used an shRNA trafficking library and screened for changes in the surface expression of CD1a antigen-presenting molecules that follow an endocytic recycling route. We found that silencing of the ADP-ribosylation factor (Arf)-like small GTPase Arl13b led to a decrease in CD1a surface expression, diminished CD1a function, and delayed CD1a recycling, suggesting that Arl13b is involved in the regulation of endocytic recycling traffic. Arl13b appears to be required for the major route of endocytic trafficking, causing clustering of early endosomes and leading to the accumulation of endocytic cargo. Moreover, Arl13b colocalized with markers of the endocytic recycling pathway followed by CD1a, namely Arf6 and Rab22a. We also detected an interaction between Arl13b and the actin cytoskeleton. Arl13b was previously implicated in cilia formation and function. Our present results indicate a previously unidentified role for Arl13b in endocytic recycling traffic and suggest a link between Arl13b function and the actin cytoskeleton.

Studies on the Bin-Amphiphysin-Rvs (BAR) domain have advanced a fundamental understanding of how proteins deform membrane. We previously showed that a BAR domain in tandem with a Pleckstrin Homology (PH domain) underlies the assembly of ACAP1 (Arfgap with Coil-coil, Ankryin repeat, and PH domain I) into an unusual lattice structure that also uncovers a new paradigm for how a BAR protein deforms membrane. Here, we initially pursued computation-based refinement of the ACAP1 lattice to identify its critical protein contacts. Simulation studies then revealed how ACAP1, which dimerizes into a symmetrical structure in solution, is recruited asymmetrically to the membrane through dynamic behavior. We also pursued electron microscopy (EM)-based structural studies, which shed further insight into the dynamic nature of the ACAP1 lattice assembly. As ACAP1 is an unconventional BAR protein, our findings broaden the understanding of the mechanistic spectrum by which proteins assemble into higher-ordered structures to achieve membrane deformation.