A cell has thousands of machines in living factories. It’s when you hear about the details that their design arouses awe and inspiration.
The Tango Trucker
German researchers just found out something interesting about how cells export cargo through their membranes. Most cargo molecules are small enough to fit into standard vesicles, but some bulky cargoes need bigger containers. According to their paper in the Proceedings of the National Academy of Sciences, a molecular machine called Tango1 (an acronym for transport and Golgi organization) “orchestrates” the handling of hefty baggage:
Exporting bulky molecules poses challenges for cells, since the membrane vesicles that transport normal-sized molecules may not be sufficiently large. The protein Tango1 allows transport vesicles to grow much larger to accommodate bulky cargo. It has been puzzling why many smaller cargos also fail to be transported when Tango1 is absent. We show that this is because bulky cargos “clog up” the transport system, resulting in a general traffic jam. Once the blocking, large cargo is removed, the jam resulting from missing Tango1 is resolved, and other cellular stress signals also subside. However, structural defects in the transport system remain, showing that these are due to a direct requirement for Tango1, independent of its function in transport as such. [Emphasis added.]
Tango1 is a large protein machine, comprised of 1907 amino acids (Cell Press). It handles the loading of large cargoes into COPII-coated vesicles (see our previous entry about vesicle coatings). The open-access PNAS paper describes what this multi-tasking protein does to grow standard pickup trucks into big rigs:
Tango1 is an ER [endoplasmic reticulum] transmembrane protein that orchestrates the loading of its cargo into vesicles by interacting with it in the ER lumen. The interaction of Tango1 with its cargo then promotes the recruitment of Sec23 and Sec24 coatomers on the cytoplasmic side, while it slows the binding of the outer layer coat proteins Sec13 and Sec31 to the budding vesicle. This delays the budding of the COPII carrier. Tango1 also recruits additional membrane material to the ERES from the Golgi intermediate compartment (ERGIC) pool, thereby allowing vesicles to grow larger. It also interacts directly with Sec16, which is proposed to enhance cargo secretion. A shorter isoform of mammalian Tango1 lacks the cargo recognition domain but nevertheless facilitates the formation of megacarrier vesicles.
A Real Pump Behind Every Human Activity
Calcium pumps are molecular motors that make muscles and nerves possible. By pumping calcium ions in and out of cell membranes, they create energy flows that get things moving. Danish scientists at Aarhus University got down close to these tiny nanomachines to watch how individual molecules in the pump actually work. Let’s first recognize their importance:
It has now become possible for the first time to look into the body’s absolutely fundamental ‘engine compartment’, and observe the ion pumps that activate cell transport and signal systems. This function ensures that you and me, and all other biological creatures on Earth, work the way they should with the right biomolecular mechanisms.
The calcium pump may not look like a lot. Each pump only measures a few nanometres, millionths of millimetres, in each direction, and sits in the cell membranes of our bodies. But despite its diminutive size, it is crucial to life. This pump is the reason that our muscles can contract, and that neurons can send signals. If the tiny pump stopped working, cells would stop communicating. Without it, we could neither move nor think. This is why cells use so much of their energy — about a fourth of the body’s fuel known as ATP — to keep the pumps running.
The team got a glimpse of this vital pump at the single-molecule level. Their work, published in Nature, reveals how the pump works as a one-way street by ensuring that ions are transported in the right direction. “The importance of such basic knowledge of biophysical processes can only be underestimated,” they comment. What they found contradicts earlier assumptions that saw the pump as a passive one-way street.
“We have identified a new closed state in the pumping cycle, which the pump can only enter if the calcium ion comes from the intracellular fluids and the pump has cleaved ATP. It cannot reach this state if the ion comes from the cell’s surroundings. When calcium is released from this state, it is the ‘point of no return’. This is the mechanism that explains that the pump works as a pump and not just a passive channel.”
This “active transport” pumping machine, powered by ATP, is so effective it can produce a 10,000-fold difference in calcium concentration from one side to the other.
Helicases are molecular motors that travel along DNA strands like railcars on a track. Different helicases can unwind DNA, duplicate DNA, and repair DNA. Another paper in PNAS reports how a team at the University of Washington improved resolution of one particular helicase by 1,000 times. “We derived a detailed mechanism for how ATP hydrolysis coordinates the motion of Hel308 along single-stranded DNA that can likely be applied to other structurally similar helicases and showed that the DNA sequence in Hel308 affects its kinetics,” they say, indicating that the information in its blueprint is crucial for its operation.
Previous studies indicated that Hel308 moves like an inchworm along a DNA strand, powered by ATP along each step. What they found was a finely-tuned interaction of energy between ADP and ATP in the movement, which guarantees directional motion:
Our data show that, instead of directly causing the conformational change, bound ATP and ADP lower the activation energy for the change, while the nucleotide’s identity (ATP or ADP) determines the direction of motion. This suggests that Hel308 walks along ssDNA [single-stranded DNA] by taking advantage of energy differences between ATP and ADP bound states, which results in directed motion of Hel308 along ssDNA.
They believe a similar process works in other helicases as well, and hope to prove it with further experiments.
The DNA Wrapper
Another machine that walks along DNA is condensin. This machine is essential for packaging DNA into chromosomes. Science Magazine reports that “The condensin complex is a mechanochemical motor that translocates along DNA.” This essential machine for DNA packing, “not been previously shown to act as a molecular motor,” not only is a motor, but really barrels down the DNA highway:
Condensin’s translocation activity is rapid and highly processive, with individual complexes traveling an average distance of ≥10 kilobases at a velocity of ~60 base pairs per second. Our results suggest that condensin may take steps comparable in length to its ~50-nanometer coiled-coil subunits, indicative of a translocation mechanism that is distinct from any reported for a DNA motor protein. The finding that condensin is a mechanochemical motor has important implications for understanding the mechanisms of chromosome organization and condensation.
A Clocklike Oscillator Controls Motion of Cilia
The title of a paper in Science Magazine indicates where this announcement is headed: “Calibrated mitotic oscillator drives motile ciliogenesis.” Know first of all that motile cilia (one of Behe’s irreducibly complex machines) create currents: “Multiciliated cells generate motile cilia-powered flows that are essential for brain, respiratory, and reproductive functions.” The “mitotic oscillator” is “a conserved clocklike regulatory circuit” involved in cell division (mitosis). A team from Paris “discovered that nondividing cells could also implement this mitotic clocklike regulatory circuit to orchestrate subcellular reorganization associated with differentiation. What was it old William Paley said about clocks?
A couple of papers shed more light on the workings of kinesin, star of one of our molecular machine animations. One paper in PNAS found that “Kinesin rotates unidirectionally and generates torque while walking on microtubules.” It sounds a bit like a happy dance; the machine does a slight twist with each step.
Given the importance of cytoskeletal motor proteins, we asked whether translational motors rotate while walking along their tracks. Using an optical tweezers-based approach, we simultaneously measured translation, force, rotation, and torque of a kinesin motor with molecular resolution. We found that the gait followed a rotary stepping mechanism that generates torque and spins cargo. Thus, during walking, the motor “tail (and organelle) will tend to wind up like the rubber band of a toy airplane,” as Joe Howard hypothesized in 1996. To determine the overall motor efficiency, our measurements also point to the importance of accounting for rotational work. Apart from other cytoskeletal motors, the technique may be applied to molecular machines such as DNA motors and rotary engines like the ATP synthase.
Another paper in PNAS reports how researchers in Houston found new insights into how kinesins cooperate. The research, summarized at Phys.org, shows that “motor proteins respond best to strong forces and hardly at all to weak ones, even those applied by motors attached to the same cargo.” It takes a lot of work to stop the kinesin up front, they found, but the “boss kinesin” doesn’t get much help from other kinesins behind, even when they are attached to the same cargo — unless they are very close together. This may facilitate the reversal of motors when they work in teams, the authors say. Whatever the reason for this “uneven force partitioning between two load-bearing kinesins,” it shows that they truly are machines using energy to perform physical work.
Close-up views of molecular machines really put the intelligence and design into ID. Consider that none of this was known just half a century ago. We’re living in exciting times for design biology. Real machines, doing real physical work in a meaningful way — it’s no wonder that all of these papers and articles are embarrassingly silent about evolution.
Illustration: From “Kinesin: The Workhorse of the Cell,” via Discovery Institute.