Technical Reference #3199

3199.

Kinase-regulated quantal assemblies and kiss-and-run recycling of caveolae Lucas Pelkmans & Marino Zerial, Max Planck Institute for Molecular Cell Biology and Genetics, Nature, 436(3199), (2005)
Link To Paper

Abstract:
A functional genomics approach has revealed that caveolae/raftmediated endocytosis is subject to regulation by a large number of kinases1. Here we explore the role of some of these kinases in caveolae dynamics. We discover that caveolae operate using principles different from classical membrane trafficking. First each caveolar coat contains a set number (one ‘quantum’) of caveolin-1 molecules. Second caveolae are either stored as in stationary multi-caveolar structures at the plasma membrane or undergo continuous cycles of fission and fusion with the plasma membrane in a small volume beneath the surface without disassembling the caveolar coat. Third a switch mechanism shifts caveolae from this localized cycle to long-range cytoplasmic transport. We have identified six kinases that regulate different steps of the caveolar cycle. Our observations reveal new principles in caveolae trafficking and suggest that the dynamic properties of caveolae and their transport competence are regulated by different kinases operating at several levels.

Materials & Methods:
Cell culture and transfection. All cells were cultured in medium containing 10% fetal calf serum. The medium for cells expressing CAV1–GFP or CAV1–mRFP contained 500 mgml21 G418. Transient transfections were performed by lipofection using FuGene6 (Roche) and short interfering RNA was transfected using Oligofectamine (Invitrogen). For imaging purposes cells were plated in glassbottomed micro-well dishes (MatTek) and maintained in CO2-independent medium(GibcoBRL). Time-lapse experimentswere performed at 25 8Cand378C. Imaging. Imaging was performed using an Olympus IX70 inverted microscope equipped with a dual-port condensor (TILL Photonics) an argon-krypton laser (Innova 70C Spectrum Coherent) and a 100Wmercury arc lamp light source to allow both Epi-FM and TIR-FM. The laser beam was focused at an off-axis position in the back focal plane of high numerical aperture £ 63/NA 1.45 (Olympus) or £ 100/NA 1.45 (Zeiss) oil immersion objectives such that the laser beam struck the interface between the glass and cell at an angle less than 558. As a result the laser light underwent total internal reflection leading to the excitation of molecules within 100nm above the interface only. For dual-colour experiments a beam splitter (Dual-View Micro-Imager Optical Insights) was used to project green and red components side by side onto a back-illuminated CCD camera (Micromax 512BFT Roper Scientific). Time-lapse sequences were acquired at 100–500-ms intervals. For high-speed imaging (17-ms intervals) a 50 £ 50 pixel region of interest was selected.

Microscopic Technique
Time-lapse

Cell Type(s)
Caveolae