Costin N. Antonescu

Advances in analysis of low signal-to-noise images link dynamin and AP2 to the functions of an endocytic checkpoint.

Numerous endocytic accessory proteins (EAPs) mediate assembly and maturation of clathrin-coated pits (CCPs) into cargo-containing vesicles. Analysis of EAP function through bulk measurement of cargo uptake has been hampered due to potential redundancy among EAPs and, as we show here, the plasticity and resilience of clathrin-mediated endocytosis (CME). Instead, EAP function is best studied by uncovering the correlation between variations in EAP association to individual CCPs and the resulting variations in maturation. However, most EAPs bind to CCPs in low numbers, making the measurement of EAP association via fused fluorescent reporters highly susceptible to detection errors. Here, we present a framework for unbiased measurement of EAP recruitment to CCPs and their direct effects on CCP dynamics. We identify dynamin and the EAP-binding α-adaptin appendage domain of the AP2 adaptor as switches in a regulated, multistep maturation process and provide direct evidence for a molecular checkpoint in CME.

Phosphatidylinositol-(4,5)-bisphosphate regulates clathrin-coated pit initiation, stabilization, and size

Phosphatidylinositol-(4,5)-bisphosphate (PIP2) is the main lipid binding partner of proteins involved in clathrin-mediated endocytosis (CME). Total internal reflection fluorescence microscopy coupled to computational image analysis revealed that the balance of PIP2 synthesis in the bulk plasma membrane and its local turnover within clathrin-coated pits control multiple distinct yet only partly overlapping stages of CME.

Phosphatidic acid plays a regulatory role in clathrin-mediated endocytosis

We have manipulated the activities of PLD and DGK, enzymes that regulate PA biosynthesis, and directly measured their effects on cellular PA levels and on clathrin-mediated endocytosis (CME). We report a previously unappreciated complexity in PA regulation and show that PA selectively regulates CME of EGF but not transferrin.

Myo1c binding to submembrane actin mediates insulin-induced tethering of GLUT4 vesicles

Insulin reduces the velocity of mobile Myo1c-positive GLUT4 vesicles beneath the muscle cell plasma membrane as visualized by total internal reflection fluorescence microscopy. Binding of vesicle-bound Myo1c to actin filaments underlies Myo1cs participation in GLUT4 vesicle tethering for subsequent productive docking and fusion of GLUT4 vesicles with the plasma membrane.

Hotspots Organize Clathrin-Mediated Endocytosis by Efficient Recruitment and Retention of Nucleating Resources

The formation of clathrin‐coated pits (CCPs) at the plasma membrane has been reported to sometimes occur repeatedly at predefined sites. However, defining such CCP ‘hotspots’ structurally and mechanistically has been difficult due to the dynamic and heterogeneous nature of CCPs. Here, we explore the molecular requirements for hotspots using a global assay of CCP dynamics. Our data confirmed that a subset of CCPs is nucleated at spatially distinct sites. The degree of clustering of nucleation events at these sites is dependent on the integrity of cortical actin, and the availability of certain resources, including the adaptor protein AP‐2 and the phospholipid PI(4,5)P2. We observe that modulation in the expression level of FCHo1 and 2, which have been reported to initiate CCPs, affects only the number of nucleations. Modulation in the expression levels of other accessory proteins, such as SNX9, affects the spatial clustering of CCPs but not the number of nucleations. On the basis of these findings, we distinguish two classes of accessory proteins in clathrin‐mediated endocytosis (CME): nucleation factors and nucleation organizers. Finally, we observe that clustering of transferrin receptors spatially randomizes pit nucleation and thus reduces the role of hotspots. On the basis of these data, we propose that hotspots are specialized cortical actin patches that organize CCP nucleations from within the cell by more efficient recruitment and/or retention of the resources required for CCP nucleation partially due to the action of nucleation organizers.

Epidermal growth factor-stimulated Akt phosphorylation requires clathrin or ErbB2 but not receptor endocytosis.

Upon ligand binding, the epidermal growth factor receptor (EGFR) activates signaling and undergoes endocytosis. EGFR signaling leading to Akt activation is impaired by perturbation of clathrin but not by inhibition of internalization through perturbation of dynamin. Clathrin may thus directly regulate receptor signaling at the cell surface.

mTOR controls lysosome tubulation and antigen presentation in macrophages and dendritic cells

LPS causes lysosome tubulation in macrophages and dendritic cells. The PI3K-Akt-mTOR pathway is necessary for LPS-induced lysosome tubulation, and mTOR is required for MHC-II presentation in dendritic cells. Evidence shows that mTOR may control lysosome tubulation by modulating microtubule motor activity through Arl8b.

Charming neighborhoods on the cell surface: Plasma membrane microdomains regulate receptor tyrosine kinase signaling

Receptor tyrosine kinases (RTK) are an important family of growth factor and hormone receptors that regulate many aspects of cellular physiology. Ligand binding by RTKs at the plasma membrane elicits activation of many signaling intermediates. The spatial and temporal regulation of RTK signaling within cells is an important determinant of receptor signaling outcome. In particular, the compartmentalization of the plasma membrane into a number of microdomains allows context-specific control of RTK signaling. Indeed various RTKs are recruited to and enriched within specific plasma membrane microdomains under various conditions, including lipid-ordered domains such as caveolae and lipid rafts, clathrin-coated structures, tetraspanin-enriched microdomains, and actin-dependent protrusive membrane microdomains such as dorsal ruffles and invadosomes. We examine the evidence for control of RTK signaling by each of these plasma membrane microdomains, as well as molecular mechanisms for how this spatial organization controls receptor signaling.

Reciprocal Regulation of Endocytosis and Metabolism

The cellular uptake of many nutrients and micronutrients governs both their cellular availability and their systemic homeostasis. The cellular rate of nutrient or ion uptake (e.g., glucose, Fe(3+), K(+)) or efflux (e.g., Na(+)) is governed by a complement of membrane transporters and receptors that show dynamic localization at both the plasma membrane and defined intracellular membrane compartments. Regulation of the rate and mechanism of endocytosis controls the amounts of these proteins on the cell surface, which in many cases determines nutrient uptake or secretion. Moreover, the metabolic action of diverse hormones is initiated upon binding to surface receptors that then undergo regulated endocytosis and show distinct signaling patterns once internalized. Here, we examine how the endocytosis of nutrient transporters and carriers as well as signaling receptors governs cellular metabolism and thereby systemic (whole-body) metabolite homeostasis.

Integrins and Cell Metabolism: An Intimate Relationship Impacting Cancer

Integrins are important regulators of cell survival, proliferation, adhesion and migration. Once activated, integrins establish a regulated link between the extracellular matrix and the cytoskeleton. Integrins have well-established functions in cancer, such as in controlling cell survival by engagement of many specific intracellular signaling pathways and in facilitating metastasis. Integrins and associated proteins are regulated by control of transcription, membrane traffic, and degradation, as well as by a number of post-translational modifications including glycosylation, allowing integrin function to be modulated to conform to various cellular needs and environmental conditions. In this review, we examine the control of integrin function by cell metabolism, and the impact of this regulation in cancer. Within this context, nutrient sufficiency or deprivation is sensed by a number of metabolic signaling pathways such as AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR) and hypoxia-inducible factor (HIF) 1, which collectively control integrin function by a number of mechanisms. Moreover, metabolic flux through specific pathways also controls integrins, such as by control of integrin glycosylation, thus impacting integrin-dependent cell adhesion and migration. Integrins also control various metabolic signals and pathways, establishing the reciprocity of this regulation. As cancer cells exhibit substantial changes in metabolism, such as a shift to aerobic glycolysis, enhanced glucose utilization and a heightened dependence on specific amino acids, the reciprocal regulation of integrins and metabolism may provide important clues for more effective treatment of various cancers.