AAGAB Controls AP2 Adaptor Assembly in Clathrin-Mediated Endocytosis

Gulbranson, Daniel R.; Crisman, Lauren; Lee, Myeongseon; Ouyang, Yu; Menasche, Bridget L.; Demmitt, Brittany A.; Wan, Chunrong; Nomura, Toshifumi; Ye, Yihong; Yu, Haijia; Shen, Jingshi

doi:10.1016/j.devcel.2019.06.013

Cited by 40 publications

(68 citation statements)

References 49 publications

(73 reference statements)

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“…Surprisingly, the transferrin receptor (TfR), a prominent receptor and known CME cargo was not among the top hits. This seems incompatible with published work and our own functional data (Figure 8) that show >5‐fold increase upon KD of AP‐2 17,27,28 . Importantly, these values, however, were obtained from quantitative fluorescence microscopy or flow cytometry using assays that probe receptor surface expression by Tf ligand binding or with anti‐receptor antibodies.…”

Section: Resultscontrasting

confidence: 90%

Inhibition of clathrin‐mediated endocytosis by knockdown of AP‐2 leads to alterations in the plasma membrane proteome

Tobys

Kowalski

Cziudaj

et al. 2020

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In eukaryotic cells, clathrin‐mediated endocytosis (CME) is a central pathway for the internalization of proteins from the cell surface, thereby contributing to the maintenance of the plasma membrane protein composition. A key component for the formation of endocytic clathrin‐coated vesicles (CCVs) is AP‐2, as it sequesters cargo membrane proteins, recruits a multitude of other endocytic factors and initiates clathrin polymerization. Here, we inhibited CME by depletion of AP‐2 and explored the consequences for the plasma membrane proteome. Quantitative analysis revealed accumulation of major constituents of the endosomal‐lysosomal system reflecting a block in retrieval by compensatory CME. The noticeable enrichment of integrins and blockage of their turnover resulted in severely impaired cell migration. Rare proteins such as the anti‐cancer drug target CA9 and tumor markers (CD73, CD164, CD302) were significantly enriched. The AP‐2 knockdown attenuated the global endocytic capacity, but clathrin‐independent entry pathways were still operating, as indicated by persistent internalization of specific membrane‐spanning and GPI‐anchored receptors (PVR, IGF1R, CD55, TNAP). We hypothesize that blocking AP‐2 function and thus inhibiting CME may be a novel approach to identify new druggable targets, or to increase their residence time at the plasma membrane, thereby increasing the probability for efficient therapeutic intervention.

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Section: Resultscontrasting

confidence: 90%

Inhibition of clathrin‐mediated endocytosis by knockdown of AP‐2 leads to alterations in the plasma membrane proteome

Tobys

Kowalski

Cziudaj

et al. 2020

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“…As recently proposed ( 86 ), this flexibility provides a mechanistic basis for the observed robustness and plasticity of CME ( 9 , 11 ). Indeed, numerous whole-genome screens for components of the endocytic machinery based on changes in cargo uptake have largely failed to identify EAPs ( 87 – 90 ). The flexible and partially redundant roles of EAPs may also explain apparent discrepancies in the literature, as the extent of involvement of a specific EAP may vary between cell types.…”

Section: Discussionmentioning

confidence: 99%

Functional characterization of 67 endocytic accessory proteins using multiparametric quantitative analysis of CCP dynamics

Bhave

Mino

Wang

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Clathrin-mediated endocytosis (CME) begins with the nucleation of clathrin assembly on the plasma membrane, followed by stabilization and growth/maturation of clathrin-coated pits (CCPs) that eventually pinch off and internalize as clathrin-coated vesicles. This highly regulated process involves a myriad of endocytic accessory proteins (EAPs), many of which are multidomain proteins that encode a wide range of biochemical activities. Although domain-specific activities of EAPs have been extensively studied, their precise stage-specific functions have been identified in only a few cases. Using single-guide RNA (sgRNA)/dCas9 and small interfering RNA (siRNA)-mediated protein knockdown, combined with an image-based analysis pipeline, we have determined the phenotypic signature of 67 EAPs throughout the maturation process of CCPs. Based on these data, we show that EAPs can be partitioned into phenotypic clusters, which differentially affect CCP maturation and dynamics. Importantly, these clusters do not correlate with functional modules based on biochemical activities. Furthermore, we discover a critical role for SNARE proteins and their adaptors during early stages of CCP nucleation and stabilization and highlight the importance of GAK throughout CCP maturation that is consistent with GAK’s multifunctional domain architecture. Together, these findings provide systematic, mechanistic insights into the plasticity and robustness of CME.

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“…Mouse preadipocytes were cultured in DMEM supplemented with 10% FB Essence (FBE, VWR, #10803‐034) and penicillin/streptomycin. The preadipocyte was a spontaneously arising immortalized cell line from mouse inguinal adipose tissue 74,75 . The cells were grown to ~95% confluence before a differentiation cocktail was added at the following concentrations: 5 μg/mL insulin (Sigma, #I0516), 1 nM Triiodo‐L‐thyronine (T3, Sigma, #T2877), 125 μM indomethacin (Sigma, #I‐7378), 5 μM dexamethasone (Sigma, #D1756), and 0.5 mM 3‐isobutyl‐1‐methylxanthine (IBMX, Sigma, #I5879).…”

Section: Methodsmentioning

confidence: 99%

“…Insulin‐stimulated GLUT4 exocytosis was measured using flow cytometry as previously described 60,75,76 . Briefly, cells were washed three times with the KRH buffer (121 mM NaCl, 4.9 mM KCl, 1.2 mM MgSO 4 , 0.33 mM CaCl 2 , and 12 mM HEPES [pH 7.4]).…”

Section: Methodsmentioning

confidence: 99%

“…B, Coomassie blue-stained SDS-PAGE gel showing recombinant NSF and α-SNAP proteins used in this study it is conceivable that tomosyn-silencing therapeutics will help unleash the full fusion capacity of GLUT4 exocytic SNAREs to improve blood glucose homeostasis in the patients.4 | MATERIALS AND METHODS4.Cell culture and adipocyte differentiationMouse preadipocytes were cultured in DMEM supplemented with 10% FB Essence (FBE, VWR, #10803-034) and penicillin/streptomycin. The preadipocyte was a spontaneously arising immortalized cell line from mouse inguinal adipose tissue 74,75. The cells were grown to 95% confluence before a differentiation cocktail was added at thefollowing concentrations: 5 μg/mL insulin (Sigma, #I0516), 1 nM Triiodo-L-thyronine (T3, Sigma, #T2877), 125 μM indomethacin (Sigma, #I-7378), 5 μM dexamethasone (Sigma, #D1756), and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma, #I5879).…”

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Genetic evidence for an inhibitory role of tomosyn in insulin‐stimulated GLUT4 exocytosis

Wang

Liu

Crisman

et al. 2020

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Exocytosis is a vesicle fusion process driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). A classic exocytic pathway is insulinstimulated translocation of the glucose transporter type 4 (GLUT4) from intracellular vesicles to the plasma membrane in adipocytes and skeletal muscles. The GLUT4 exocytic pathway plays a central role in maintaining blood glucose homeostasis and is compromised in insulin resistance and type 2 diabetes. A candidate regulator of GLUT4 exocytosis is tomosyn, a soluble protein expressed in adipocytes. Tomosyn directly binds to GLUT4 exocytic SNAREs in vitro but its role in GLUT4 exocytosis was unknown. In this work, we used CRISPR-Cas9 genome editing to delete the two tomosyn-encoding genes in adipocytes. We observed that both basal and insulinstimulated GLUT4 exocytosis was markedly elevated in the double knockout (DKO) cells. By contrast, adipocyte differentiation and insulin signaling remained intact in the DKO adipocytes. In a reconstituted liposome fusion assay, tomosyn inhibited all the SNARE complexes underlying GLUT4 exocytosis. The inhibitory activity of tomosyn was relieved by NSF and α-SNAP, which act in concert to remove tomosyn from GLUT4 exocytic SNAREs. Together, these studies revealed an inhibitory role for tomosyn in insulin-stimulated GLUT4 exocytosis in adipocytes. We suggest that tomosyn-arrested SNAREs represent a reservoir of fusion capacity that could be harnessed to treat patients with insulin resistance and type 2 diabetes.

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AAGAB Controls AP2 Adaptor Assembly in Clathrin-Mediated Endocytosis

Cited by 40 publications

References 49 publications

Inhibition of clathrin‐mediated endocytosis by knockdown of AP‐2 leads to alterations in the plasma membrane proteome

Inhibition of clathrin‐mediated endocytosis by knockdown of AP‐2 leads to alterations in the plasma membrane proteome

Functional characterization of 67 endocytic accessory proteins using multiparametric quantitative analysis of CCP dynamics

Genetic evidence for an inhibitory role of tomosyn in insulin‐stimulated GLUT4 exocytosis

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