One of the most fascinating features of biology is the movement of organisms, subcellular compartments, even single proteins and ions. While high organisms use muscle contraction for motion, the way things move inside the cell is simply carried out by diffusion of the material inside the cytoplasm. Enzymes can therefore find their targeted molecule or location by random Brownian motion. However, life can be more demanding than random diffusion in certain situations and the cell needs different means of transport to move organelles, chromosomes, sensors and other enzymes in a directed manner. This process requires driving mechanical energy that is compensated by utilizing the chemical energy driven by ATP hydrolysis. For example, duplicated chromosomes can move to the metaphase plate before cell division by rearranging the intracellular formation of microtubule filaments through polymerization. Synapses and dendrites can be far from the cell body of neurons and required receptor molecules and organelles are transported along the axons by the help of molecular motors moving along the microtubule.
The proteins that can transduce the chemical energy to force and movement are called motor proteins. Single molecule biophysics is an emerging field to study fundamental biological processes carried out by single motors. By measuring subnanometer movements and picoNewton levels of forces, biophysical techniques have elucidated new aspects of how proteins and nucleic acids work individually and how their collective behavior affects cell’s function.
My research is focused on pushing the limits of sensitivity of microscopes to study individual microtubule- and DNA-based enzymes. We develop single molecule fluorescence and force clamp techniques to understand the structural basis of how motor proteins can generate force and how they can carry a cargo 10.000 fold larger than its size over long distances. I am also interested in super-resolution imaging of chromosome structure and multiprotein complexes in live cells.
The projects in the lab combine techniques in physics, molecular biology and biochemistry to understand the mechanism of complex enzymatic machinery in vitro and in living cells. Major focus is on cytoplasmic dynein motor, intracellular cargo transport, and chromosome end protection.
Dissecting the Molecular Mechanism of Dynein: Cytoplasmic dynein is a unique motor that transports a variety of intracellular cargo towards the microtubule minus-end in eukaryotic cells. However, a structural dissection of its mechanism has not been undertaken. By using budding yeast cells to genetically manipulate and express the dynein motor, we perform single molecule FRET experiments to measure interactions between the internal domains in an active protein and investigate how these interactions are correlated with the movement of the motor. The locations of the rings and their rotations will be detected by single molecule polarization microscopy as dynein walks along microtubules. These experiments will help us to draw a detailed mechanistic model of how dynein works in cells.
Single Molecule Studies on Intraflagellar Transport (IFT): To elucidate how cargoes are transported in vivo, my lab is using Chlamydomonas cells as a model system. So far, we have tracked flagellar membrane proteins and observed that these proteins are transported uniformly toward a single direction. By using this system, we aim to understand how opposite polarity motors function together to move cargoes back and forth along the microtubules and how cells control their activity by associated enzymes. We use mutated strains of Chlamydomonas to externally control motor activity and manipulate cargo transport by applying forces via optical tweezers. Our studies will also reveal how cells can rapidly grow and maintain cilia and flagella by the help of these measurements.
Telomere Loop Formation: Telomeric DNA repeats protect ends of linear chromosomes against degradation. Because of the “3’-end replication problem”, telomeres constantly shorten upon each cell division and critically short telomeres lead to cell cycle arrest. Both aging and human cancers have been related to this important system. Although telomeres and telomere binding proteins have been extensively studied by genetic manipulations and biochemical methods, understanding the formation of functional telomere and its interaction with telomerase and other binding partners needs more sensitive measurements. We aim to develop a single molecule assay by reconstituting human telomere complex in vitro to observe how telomeres form loops for end capping of chromosomes and how these loops open and close as a replication fork reaches to the telomere.
Ahmet Yildiz, Michio Tomishige, Arne Gennerich and Ronald D. Vale. Intramolecular strain governs kinesin stepping behavior along microtubules. 2008. Cell (in press).
Samara L. Reck-Peterson, Ahmet Yildiz, Andrew P. Carter, Arne Gennerich, Nan Zhang, Ronald D. Vale. Cytoplasmic dynein coordinates its two motor domains to move processively along microtubules. 2006. Cell 126, 335.
Ahmet Yildiz. How Molecular Motors Move. 2006. Science 311, 792.
Ahmet Yildiz, Michio Tomishige, Ronald D. Vale, Paul R. Selvin. Kinesin walks hand-over-hand. 2004. Science 303, 676.
Ahmet Yildiz, Joseph N. Forkey, Sean A. McKinney, Taekjip Ha, Yale E. Goldman, Paul R. Selvin. Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5 nm localization. 2003. Science 300, 2061.