Dual control of caveolar membrane traffic by microtubules and the actin cytoskeleton

Abstract

Live cell, time-lapse microscopy was used to study trafficking of caveolin-1-GFP in stably expressing CHO cells. Multiple cytological and biochemical tests verified that caveolin-1-GFP was a reliable marker for endogenous caveolin-1. At steady state, most caveolin-1-GFP was either at the cell surface associated with invaginated caveolae or near the centrosome in caveosomes. Live cell fluorescence imaging indicated that while much of the caveolin-1-GFP in caveolae at the cell surface was relatively sessile, numerous, highly motile caveolin-1-GFP-positive vesicles were present within the cell interior. These vesicles moved at speeds ranging from 0.3-2 μm/second and movement was abolished when microtubules were depolymerized with nocodazole. In the absence of microtubules, cell surface invaginated caveolae increased more than twofold and they became organized into linear arrays. Complete depolymerization of the actin cytoskeleton with latrunculin A, by contrast, triggered rapid and massive movements of caveolin-positive structures towards the centrosomal region of the cell. The caveolar membrane system of CHO cells therefore appears to be comprised of three caveolin-1-containing compartments. These include caveolae that are confined to the cell surface by cortical actin filaments, the peri-centrosomal caveosomes and caveolar vesicles, which we call `cavicles', that move constitutively and bi-directionally along microtubules between the cell surface and caveosomes. The behavior of cavicles suggests that they function as transport intermediates between caveolae and caveosomes.

HPV-16 E5 oncoprotein upregulates lipid raft components caveolin-1 and ganglioside GM1 at the plasma membrane of cervical cells

“…Treatment of HECs with latrunculin A, a potent, membrane-permeable anti-microfilament agent, does completely depolymerize microfilaments in all three HEC lines (Figure 7a). Since microtubules also influence the distribution of caveolin-1 between the plasma membrane and Golgi apparatus in vivo (Mundy et al, 2002), we also determined that 16E5 does not disrupt microtubules in HECs (Figure 7a).…”

Intracellular trafficking of nonviral vectors

“…Mundy et al 79 showed that caveolae were retained on the cell surface by cortical actin filaments (Figure 2). Disruption of actin by lantrunculin A facilitated the pinching off and internalization of caveolar vesicles, or 'cavicles', which then trafficked along microtubules to pericentrisomal caveosomes.…”

Cholesterol-sensitive Modulation of Transcytosis

“…Although TfnRs are canonically thought to be excluded from DRMs, recent evidence seems to support a possible link between TfnRs and lipid rafts: 1) recycling endosomes enriched with Tfn immunoisolated from MDCK cells contained caveolins and other raft components (Gagescu et al, 2000); 2) the folate receptor (a glycosylphosphatidylinositollinked protein), which typically clusters in caveolae, has been found in endosomes containing Tfn (Mayor et al, 1998); 3) recycling endosomes rich in TfnRs were localized in proximity to centrosome-associated caveosomes (Maxfield and Yamashiro, 1987;Mundy et al, 2002); and 4) a fraction of TfnRs recruited under certain conditions into immunological synapses was reported to be associated with DRMs (Batista et al, 2004).…”

“…They also demonstrated that Cav1 is phosphorylated on tyrosine 14 (pY14-Cav1) by the oncogenic v-Src kinase (Li et al, 1996) and by normal cellular Src kinases (Lee et al, 2001;Kim et al, 2002), including c-Yes (Lee et al, 2001), a kinase previously shown to be involved in the stimulation of dIgA transcytosis . The proximity between intracellular caveolar structures and recycling endosomes (Gagescu et al, 2000;Mundy et al, 2002) led us to assume that a cross-talk between postendocytic membrane traffic pathways and intracellular Cav1 may be established in cholesterol-depleted cells. The result of this process could be the overall facilitation of signal transduction pathways relevant for stimulation of transcytosis.…”