Abstract
The pacsin (also termed syndapin) protein family is well characterised structurally. They contain F-BAR domains associated with the generation or maintenance of membrane curvature. The cell biology of these proteins remains less understood. Here, we initially confirm that EHD2, a protein previously shown biochemically to be present in caveolar fractions and to bind to pacsins, is a caveolar protein. We go on to report that GFP-pacsin 2 can be recruited to caveolae, and that endogenous pacsin 2 partially colocalises with caveolin 1 at the plasma membrane. Analysis of the role of pacsin 2 in caveolar biogenesis using small interfering RNA (siRNA) reveals that loss of pacsin 2 function results in loss of morphologically defined caveolae and accumulation of caveolin proteins within the plasma membrane. Overexpression of the F-BAR domain of pacsin 2 (but not the related F-BAR domains of CIP4 and FBP17) disrupts caveolar morphogenesis or trafficking, implying that pacsin 2 interacts with components required for these processes. We propose that pacsin 2 has an important role in the formation of plasma membrane caveolae.
Highlights
Caveolae are flask-shaped invaginations in the plasma membranes of many mammalian cell types, where they have several important functions in diverse processes, including transcytosis, metabolic regulation, clathrin-independent endocytosis and signalling (Doherty and McMahon, 2009; Hansen and Nichols, 2009; Lajoie and Nabi, 2010; Le Lay and Kurzchalia, 2005; Parton and Simons, 2007; Pilch et al, 2007; van Deurs et al, 2003)
EHD2 and Pacsin 2 can be recruited to caveolae In order to confirm that EHD2 can be recruited to caveolae, we expressed GFP–EHD2 in HeLa cells and labelled caveolin 1 using indirect immunofluorescence
The membrane puncta colocalised with caveolin 1, with over 75% of GFP–EHD2 puncta containing caveolin 1
Summary
Caveolae are flask-shaped invaginations in the plasma membranes of many mammalian cell types, where they have several important functions in diverse processes, including transcytosis, metabolic regulation, clathrin-independent endocytosis and signalling (Doherty and McMahon, 2009; Hansen and Nichols, 2009; Lajoie and Nabi, 2010; Le Lay and Kurzchalia, 2005; Parton and Simons, 2007; Pilch et al, 2007; van Deurs et al, 2003). Caveolins 1 and 2 are both multiply acylated (Dietzen et al, 1995; Monier et al, 1996) and have a stretch of hydrophobic amino acids that is likely to be embedded within the lipid bilayer They hetero-oligomerise in the Golgi complex to form caveolin microdomains (Monier et al, 1995; Parolini et al, 1999) and travel together en route to the plasma membrane (Hayer et al, 2010a; Tagawa et al, 2005), where they associate with the cavins (Bastiani et al, 2009; Hansen et al, 2009; Hansen and Nichols, 2010; Hill et al, 2008; Liu and Pilch, 2008; McMahon et al, 2009; Vinten et al, 2005). The key step in caveolar biogenesis, in which flat caveolin microdomains combine with cavins and potentially additional proteins to produce the characteristic caveolar membrane morphology, is still not fully understood
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