Loss of PTEN promotes formation of signaling-capable clathrin-coated pits

Abstract

Defective endocytosis and vesicular trafficking of signaling receptors has recently emerged as a multifaceted hallmark of malignant cells. Clathrin-coated pits (CCPs), the fundamental unit of clathrin-mediated endocytosis, display highly heterogeneous dynamics on the plasma membrane where they can take from 20 seconds to over a minute to form cytosolic coated-vesicles. Despite the large number of cargo molecules that traffic through CCPs, it is not well understood whether signaling receptors activated in cancer, such as epidermal growth factor receptor (EGFR), are regulated through a specific subset of CCPs. The signaling lipid phosphatidylinositol (3,4,5)-triphosphate (PI(3,4,5)P 3 ), which is dephosphorylated by phosphatase tensin homolog (PTEN), is a potent tumorigenic signaling lipid that is present in excess in many types of cancers. Using total internal reflection fluorescence microscopy and automated tracking and detection of CCPs, we find PTEN and EGF bound EGFR are enriched in a distinct subset of short-lived CCPs that corresponded with clathrin-dependent EGF-induced signaling. By deleting PTEN using CRISPR-Cas9 and reconstituting PTEN, we demonstrate that PTEN plays a role in the regulation of CCP dynamics; this appears to recapitulate CCP dynamics in highly metastatic PTEN-deleted cancer cells where we find a larger proportion of short-lived CCPs and higher initiation density compared to the normal cells. Furthermore, increased PI(3,4,5)P 3 results in higher proportion of short-lived CCPs, an effect that recapitulates PTEN deletion. Our findings provide strong evidence for the existence of short-lived 'signaling-capable' CCPs. Altogether, these findings demonstrate the importance of PTEN and PI(3,4,5)P 3 in regulating CCP dynamics and assign a new function to PTEN as a modulator of signaling-capable CCPs.peer-reviewed)

Complimentary action of structured and unstructured domains of epsin supports clathrin-mediated endocytosis at high tension

“…Previous work from our lab have shown that retinal pigment epithelial (RPE) cells spread on large fibronectin islands (to induce high membrane tension), exhibited an increase in proportion of abortive CCSs and smaller CCPs 22,23 . Using SIM-TIRF super-resolution imaging of RPE cells stably expressing mCherry-clathrin light chain (CLC), we characterized the morphology of CCSs and classified them into three categories: (i) Abortive CCSs, which are coated structures which dissemble before they reach maturation [24][25][26] , (ii) Stalled CCSs, which are persistent, noninternalizing coated structures in the imaging field 8,12 , and (iii) Productive CCPs, which are coated structures undergo initiation, assembly, and transition to coated-pits followed by membrane scission and internalization. Lifetime analyses of CCSs have shown that abortive CCSs have a lifetime less than 20 s 27-29 and stalled CCSs have a lifetime above 120 s 8,12,27 .…”

The big and intricate dreams of little organelles: Embracing complexity in the study of membrane traffic

“…This has revealed broad heterogeneity of CCPs, including in size, distribution within the cell surface, lifetime and protein composition. 48,70,[76][77][78][79][80][81][82][83][84][85][86] In addition, specific CCP properties have been linked to cargo receptor content. 78,85 This type of data has allowed the construction of computational models to describe CCP assembly and scission, 87 from which now emerges the synergism between predictions made by these computational models and experimental testing that are at the core of systems biology.…”

PTENα regulates endocytosis and modulates olfactory function

“…Recently, PTEN is also shown to play a role in regulation of CCP dynamics. PTEN regulates the proportion of shortlived CCPs depending on its lipid phosphatase activity (49). According to our study, PTENa is involved in CME regulation by interacting with amphiphysin depending on its protein phosphatase activity.…”