Spring-Summer 2007
The Role of Kinesin and Dynein on Membrane Protein Transport in Intraflagellar Transport
Ahmet Yildiz, Fatih Kocabas, Wallace D. Marshall, Ronald D. Vale
Department of Cellular and Molecular Pharmacology, University of California, San Francisco
Dynein and kinesin motors are responsible for all minus- and plus-end directed transport along microtubules in cells, respectively. To understand how they transport cargos within the cell, we use the membrane protein transport on Chlamydomonas flagella as a model system. Flagellum lacks protein synthesis machinery, and axonemal precursors and sensory membrane proteins are moved continuously from the cell body towards the tip of the flagella by intraflagellar transport. (IFT). At the tip of the flagellum, IFT reverses the direction and moves back to the basal body to restart the transport process. We have attached a fluorescently labeled beads to a flagellar glycoprotein, FMG-1, and monitored its transport. We have observed that membrane proteins move in a stepwise fashion, indicative of motor proteins carrying the cargo. Kinesin and dynein motors coordinately move the cargo without showing frequent back and forth movement in a short time scale. This observation differs significantly from the bidirectional transport on neuronal axons. In IFT, kinesin and dynein motors are highly coordinated in a way that they work without competing with each other, presumably one motor is active and the other is inactive. Elucidation of how the cell achieves this organization requires future single molecule biophysical work.
Chlamydomonas Reinhardtii and Intraflagellar Transport (IFT)
Chlamydomonas is a single cell alga that moves by twitching its two flagella.Immunofluorescence of IFT particle shows that they are localized primarily in the base of flagella and there is only a few moving IFT particles along the axonemes.
The directed movement of IFT particles is most likely provided by microtubule-based motors. Recent studies on kinesin and dynein deleted mutants in Chlamydomonas approved the role of motor proteins. Particles were observed to move anterogradely (from cell body to the tip) at 2.5 mm/sec and retrogradely at 4 mm/sec.
Flagella are formed by an axoneme surrounded by a flagellar membrane. To grow and maintain flagella, particles are moved back and forth along axonemes by intraflagellar transport (IFT). This process is an essential mechanism and defects can be primary causes of some human diseases including polycystic kidney disease and retinal degeneration.
Paralyzed-flagella (pf) mutants of Chlamydomonas were completely immobilized on a polylysine coated glass surface. Fluorescent beads covalently linked to FMG-1 monoclonal antibody bind to flagellar surface and show unidirectional movement along the flagella.
CHLAMY LAB NOTES>>
Bu projede hucre zari ve kamci uzerinde proteinlerin nasil tasindigini "tek molekule takip" metoduyla anlamaya calistik. Bildigimiz kadariyla bazi bobrek hastaliklar kamci ile ilgili kamci ici tasinimin sorunlu olmasindan kaynaklaniyor, ve bazen bu gelisim doneminde baska hastaliklara da neden olabiliyor. Calisamda en son teknolojiyi kullanarak daha hizli kameralarla bir tek parlak molekulu zardaki proteinlere antikolarin baglanmasiyla zardaki protein tasinmasini takip ediyoruz.