Rice University researchers have engineered cells to characterize how sensitively altering the cooperative functions of motor proteins can regulate the transport of organelles.
Scientists at Rice University regulated the size of vesicular organelles called peroxisomes and the density of protein motors on their surfaces to analyze how the motors cooperate as they move the vesicles inside cells. The PEX 3/FKBP receptors, when incubated with rapalog, allow transiently expressed kinesin and myosinVa motors to be coupled to vesicle surfaces via FRB protein fusions. Once coupled to the vesicles, the motors pull them along cytoskeleton filaments (bottom). (Credit: Diehl Lab/Rice University)
The study, by the Rice lab of bioengineer and chemist Michael Diehl, compared the collective behaviors of kinesin-1 and myosinVa in living cells to determine how these motor proteins cooperate as they move vesicles and organelles along intracellular highways formed from cytoskeletal filaments. These transport processes are critical to numerous developmental and signaling functions within cells, and breakdowns in motor functions are also implicated in several human diseases.
The work appears this week in the Proceedings of the National Academy of Sciences.
Diehl and his colleagues at Rice’s BioScience Research Collaborative, including Rice postdoctoral researchers Anand Radhakrishan and Artem Efremov and graduate student David Tsao, compared the collective responses of the motor proteins to variations in motor numbers and cargo sizes.
They began with a good understanding of the collective pulling power of kinesin motors. Kinesin is a type of protein that binds to and transports cargoes by walking along cytoskeletal filaments called “microtubules.” In previous experiments, they engineered multiple motor systems that were anchored to polystyrene beads as an experimental cargo, but this time decided to engineer organelles called “peroxisomes” within living cells for these analyses.
“Our earlier work was detailed and very precise, but the central limitation was simply that the motors were not transporting a real cargo,” Diehl said. In contrast to rigid beads, many organelles like peroxisomes have fluid-like lipid membranes. Motors attached to their surfaces will therefore interact in ways that are difficult to recapitulate using plastic beads.
“The physical environment inside a living cell is also difficult to emulate during in-vitro experiments,” he said. “This could also have an appreciable impact on how motors cooperate to transport their cargoes.”
Using genetically engineered COS cells, the team coupled motor proteins to peroxisomes via a certain type of protein switch. They regulated the expression of these protein switch genes in combination with a second gene that allowed them to tune the final density of motors on the surfaces of peroxisomes as well as the distributions of peroxisome sizes.
The genetic-level controls allowed the group to evaluate how the collective behaviors of kinesin and myosinVa motors responded to changes in motor levels and size-dependent forces imposed on the peroxisomes by the cytoplasm. In contrast to behaviors found for kinesins, they show that the cargoes move more rapidly when myosinVa levels are altered and that myosin systems are more readily capable of producing the forces necessary to propel large cargos in living cells.
“Kinesins are like racehorses that basically only know how to run fast,” Diehl said. “They don’t like to work together in teams to transport cargos, and usually one kinesin is left doing the work of transporting a cargo on its own. MyosinVa motors, however, are more like stagecoach horses. If you add more horses, you get more force production. You get more speed. This distinction is important, since it suggests that collective functions of myosinVa can be regulated more sensitively than kinesins.”
Kinesin and myosinVa are bound simultaneously to the surfaces of many organelles, Diehl said. The collective force-producing capacities and responses of cargo transport to variation in motor copy number will influence the trafficking of cargoes to different regions of cells. “Cells needs to be able to regulate that competition, and so it may be useful to have one motor, kinesin, that’s strong individually but not capable of cooperating, and another motor that might be weak individually but have a strong cooperative effect,” he said. “It means all the fine tuning can take place on one side.”
The overall results open a new window into cellular mechanisms, Diehl said. “Now that we’re able to probe detailed relationships between motor type, ratio, cargo size and force, we can start to examine more complicated collective and regulatory behaviors directly,” he said. “We hope to recapitulate scenarios where multiple normal motors are bound to the surfaces of cargoes simultaneously with motor mutants that have been associated with neurodegenerative diseases. This way, we can precisely examine how these mutants perturb intracellular transport pathways.”
Co-authors of the paper include Carol Bookwalter, a staff member, and Kathleen Trybus, a professor of molecular physiology and biophysics at the University of Vermont.
The Welch Foundation and the National Institutes of Health supported the research.
Jeff Falk | EurekAlert!
Symbiotic bacteria: from hitchhiker to beetle bodyguard
28.04.2017 | Johannes Gutenberg-Universität Mainz
Nose2Brain – Better Therapy for Multiple Sclerosis
28.04.2017 | Fraunhofer-Institut für Grenzflächen- und Bioverfahrenstechnik IGB
More and more automobile companies are focusing on body parts made of carbon fiber reinforced plastics (CFRP). However, manufacturing and repair costs must be further reduced in order to make CFRP more economical in use. Together with the Volkswagen AG and five other partners in the project HolQueSt 3D, the Laser Zentrum Hannover e.V. (LZH) has developed laser processes for the automatic trimming, drilling and repair of three-dimensional components.
Automated manufacturing processes are the basis for ultimately establishing the series production of CFRP components. In the project HolQueSt 3D, the LZH has...
Reflecting the structure of composites found in nature and the ancient world, researchers at the University of Illinois at Urbana-Champaign have synthesized thin carbon nanotube (CNT) textiles that exhibit both high electrical conductivity and a level of toughness that is about fifty times higher than copper films, currently used in electronics.
"The structural robustness of thin metal films has significant importance for the reliable operation of smart skin and flexible electronics including...
The nearby, giant radio galaxy M87 hosts a supermassive black hole (BH) and is well-known for its bright jet dominating the spectrum over ten orders of magnitude in frequency. Due to its proximity, jet prominence, and the large black hole mass, M87 is the best laboratory for investigating the formation, acceleration, and collimation of relativistic jets. A research team led by Silke Britzen from the Max Planck Institute for Radio Astronomy in Bonn, Germany, has found strong indication for turbulent processes connecting the accretion disk and the jet of that galaxy providing insights into the longstanding problem of the origin of astrophysical jets.
Supermassive black holes form some of the most enigmatic phenomena in astrophysics. Their enormous energy output is supposed to be generated by the...
The probability to find a certain number of photons inside a laser pulse usually corresponds to a classical distribution of independent events, the so-called...
Microprocessors based on atomically thin materials hold the promise of the evolution of traditional processors as well as new applications in the field of flexible electronics. Now, a TU Wien research team led by Thomas Müller has made a breakthrough in this field as part of an ongoing research project.
Two-dimensional materials, or 2D materials for short, are extremely versatile, although – or often more precisely because – they are made up of just one or a...
28.04.2017 | Event News
20.04.2017 | Event News
18.04.2017 | Event News
28.04.2017 | Medical Engineering
28.04.2017 | Earth Sciences
28.04.2017 | Life Sciences