Molecular machines visualized in 3D
Cryo-electron microscopy allows cell biologists to see irreducibly complex molecular machines in all their three-dimensional glory. Today, we are privileged to see things that earlier microscopists could not have dreamed were possible with super-resolution imaging technologies.
Molecular biologists from the University of Basel boasted this month of having discovered a “miniature train station” at the base of the cilium. Evolution News readers may know that biologist Michael Behe discussed these trains years ago in his first two books, based on what was then known about cilia construction. A few attempts to animate cilia with earlier cryo-EM views, such as this one at XVIVO, reveal the parts of cilia and flagella and how they function once constructed. But there’s nothing like imaging them with real microscopes at near atomic resolution to see how they’re constructed. The station imagery seems appropriate.
Cilia are firmly anchored to the cell at their base. “Here is the starting station for eyelash transport,” says Hugo van den Hoek, first author of the study. “The trains are assembled here, loaded with goods and placed on the tracks.”There are in total nine different tracks inside the cilia, called microtubules. Each of them consists of two tracks, one for outgoing trains and one for incoming trains. Trains carry proteins such as signaling molecules and building materials to the end of the eyelashes. At their destination station, the train is unloaded and disassembled. [Emphasis added.]
A combination of cryo-EM tomography and fluorescence microscopy allowed the research team to observe the large central station.
“Thanks to fluorescence microscopy, we also know the exact schedule trains. The trains leave the departure station within nine seconds, and then the whole process of assembling the train begins again.
An additional method called Expansion Microscopy mapped all parts on tomography data.
“This powerful combination of technologies made it possible to reconstruct the first molecular model of the ciliary base and to observe how it regulates the assembly and entry of these large protein trains“, explains Paul Guichard.
Does anyone have any sense of foresight here? Functional information?
The paper in Science by Van den Hoek et al., “In situ architecture of the ciliary base reveals stepwise assembly of intraflagellar transport trains”, continues the train metaphor more than 90 times.
Illustrating Behe’s 1996 claim that scientific papers never explain how these machines could be made by a Darwinian process, this paper is again silent on evolution. He only notes that cilia and flagella are “evolutionarily conserved eukaryotic organelles”, implying that they once seemed to function and have not evolved significantly since. They also mention serious illnesses resulting from faulty assembly of these exquisite mobile ATP-powered machines. This also testifies to the impossibility of the formation of chance.
Readers can feast on detailed images from these powerful new imaging technologies.
“Their findings explain how intraflagellar transport trains come together before they enter the eyelashes and demonstrate the possibility of visualize dynamic events with molecular resolution inside native cells.”
The eyelashes were also highlighted recently in news from the University of Washington. Engineers there would like to understand how cilia initiate their well-known beating motions to get ideas for treating ciliopathies and, perhaps, mimicking cilia in machines designed for drug delivery or chemical sensing.
Cilia are tiny hair-like structures on cells all over our body that beat rhythmically to serve a variety of functions when working properlyincluding the circulation of cerebrospinal fluid in the brain and the transport of eggs through the fallopian tubes.
Defective eyelashes can lead to disorders including situs inversus – a condition in which a person’s organs grow on the opposite side to where they usually are.
Their work has been published in the Jjournal of the Royal Society Interface.
Kir2.1: an elegant ion channel
Cryo-electron microscopy has unveiled another marvelous molecular machine in the eyes of Sorbonne researchers. It’s called Kir2.1, which is part of a family of potassium channels that create the voltage used by neurons. Here is what Kir2.1 does for us:
Internally rectifying (Kir) potassium channels are a group of integral membrane proteins this selectively control K permeation+ ions (potassium) through cell membranes. They have the particularity that the channels K-pipe+ions more easily inward (into the cell) than outward (outside the cell). The little outdoor K+ current through Kir channels controls resting membrane potential and membrane excitability, regulates cardiac and neural electrical activitiescouples insulin secretion to blood glucose levels, and maintains electrolyte balance.
The source article from Fernandes et al. has been published in open access in Scientists progress allowing readers to see the beautiful images of this channel with its four-part structure and selectivity filter. They claim that this is the “first published structure” of Kir2.1. The average resolution is 4.3 Angstroms, with some parts at 3.7 Angstroms. Considering that the width of a hydrogen atom is about 1 Angstrom, that’s amazing.
It’s the first time that the entire Kir2.1 human channel was resolved at high resolution; it is also the first cryo-EM structure of a Kir2 channel.
How does the channel work as a rectifier, creating tension between the inner and outer membranes? And how do they know when to act?
The inward grinding mechanism results from a blockage on the cytoplasmic face of the channels by endogenous polyamines and Mg2+ which plug the channel pore at depolarized potentialsleading to a decrease in outgoing currents. The blockers are then removed from the pore when the K+ ions enter the cell at hyperpolarized potentials. This voltage dependent block results in effective current conduction only inwards. In addition to internally rectifying, Kir channels respond to a variety of intracellular messengers that directly control channel gating, including phosphoinositides (PIPs), G-proteins (Kir3 channels), adenosine 5′-triphosphate (Kir6 channels), and changes in pH (Kir1 channels). The Kir family is encoded by 16 genes (KCNJ1 to KCNJ18) and classified into seven subfamilies (Kir1 to Kir7).
The Kir2.1 channel doesn’t just sit there in the membrane selecting potassium ions; it moves! It flexes and bends during operation. Readers can download six films of the machine undergoing its precise conformational changes.
As the channel flexes, specific contacts between amino acids are made and broken to allow the precise passage of potassium ions through the selectivity filter and three other points of constriction, one called the G-loop where the final potassium trigger is believed to occur. The constrictions, as narrow as 1/5 of an Angstrom, act like gates blocking everything until the correct potassium ion has been authenticated. Here’s a taste of the precision:
In conclusion, our human Kir2.1 channel cryo-EM structure describes a well-connected interaction networkbetween the PIP2 binding site residues, R218 and K219, and the G-loop region (E303) via residues R312 and H221. Our data suggest that the conformational changes required for G-loop opening are most likely controlled via PIP2 link. The replacing R312 with histidine results in complete loss of the interaction network described above. Therefore, the interaction network integrity between the subunits seems necessary for the good allosteric transmission of the signal between R312 and the G-loop of the adjacent subunit during PIP2 binding, which maybe allows the release of the point of constriction on the loop G. We can then put forward the hypothesis of a loop G dependent on PIP2 trigger mechanism which consists of the following elements: the PIP2 link triggers conformational changes in the position of the side and main chains of R218 and K219, which, due to the structural proximity, lead to significant changes in the position of H221, moving it laterally towards the intracellular medium. This move would in turn cause E303 and R312 of the adjacent chain to move in the same direction, causing the loop G to open.
Without wishing to abuse technical jargon, the design only imposes itself in the details. Once again, readers will search in vain for any mention of the emergence or evolution of this chain.