Grand Central Station and Beyond: Molecular Machines Visualized in 3-D
Cryo-electron microscopy is allowing cell biologists to see irreducibly complex molecular machines in all their three-dimensional glory. We are privileged in our day to see things that earlier microscopists could not have dreamt were possible thanks to super-resolution imaging technologies.
Molecular biologists at the University of Basel boasted this month about discovering a “miniature train station” at the base of the cilium. Evolution News readers may know that biologist Michael Behe spoke of these trains years ago in his first two books, based on what was then known about how cilia are constructed. 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 operate once constructed. But there’s nothing like imaging them with real microscopes at near-atomic resolution to see how they are built. The imagery of train stations seems appropriate.
Cilia are firmly anchored to the cell at their base. “Here is the start station for cilia transport,” explains Hugo van den Hoek, first author of the study. “Trains are assembled here, loaded with cargo and placed on the rails.”There are a total of nine different rails inside cilia, called microtubules. Each of them consists of two tracks, one for outbound trains and one for inbound. The trains transport proteins such as signaling molecules and building materials to the tip of the cilia. 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 grand central station.
“From fluorescence microscopy, we also know the exact timetable of the trains. Trains leave the start station within nine seconds, and then the whole train assembly process starts again.”
An additional method called Expansion Microscopy allowed mapping of all the parts on the tomography data.
“This powerful combination of technologies has allowed us to reconstruct the first molecular model of the ciliary base and observe how it regulates the assembly and entry of these large protein trains,” explains Paul Guichard.
Anyone sense foresight here? Functional information?
The paper in Science by Van den Hoek et al., “In situ architecture of the ciliary base reveals the stepwise assembly of intraflagellar transport trains,” continues with the train metaphor over 90 times.
Illustrating Behe’s claim back in 1996 that scientific papers never explain how these machines could be made by a Darwinian process, this paper is again silent about evolution. It only notes that cilia and flagella are “evolutionarily conserved eukaryotic organelles” which implies that they appeared already working and have not evolved significantly since. They also mention serious diseases that result from faulty assembly of these exquisite ATP-powered moving machines. This also speaks to the impossibility of chance formation.
Readers can feast their eyes on the detailed images coming from these powerful new imaging technologies.
“Their findings elucidate how intraflagellar transport trains assemble before they enter cilia and demonstrate the possibility of visualizing dynamic events with molecular resolution inside native cells.”
Cilia were also highlighted recently in news from Washington University. Engineers there would like to understand how cilia initiate their well-known beat motions to get ideas for treating ciliopathies and, perhaps, mimicking cilia in engineered machines for drug delivery or chemical sensing.
Cilia are tiny, hair-like structures on cells throughout our bodies that beat rhythmically to serve a variety of functions when they are working properly, including circulating cerebrospinal fluid in brains and transporting eggs in fallopian tubes.
Defective cilia can lead to disorders including situs inversus — a condition where a person’s organs develop on the side opposite of where they usually are.
Their work was published in the Journal of the Royal Society Interface.
Kir2.1: An Elegant Ion Channel
Cryo-electron microscopy unveiled another marvelous molecular machine to the eyes of researchers at the Sorbonne. It’s called Kir2.1, part of a family of potassium channels that create the voltage used by neurons. Here’s what Kir2.1 does for us:
Inward-rectifier potassium (Kir) channels are a group of integral membrane proteins that selectively control the permeation of K+ (potassium) ions across cell membranes. They are particular in that the channels conduct K+ions easier in the inward direction (into the cell) than in the outward direction (out of the cell). The small outward K+ current through Kir channels controls the resting membrane potential and membrane excitability, regulates cardiac and neuronal electrical activities, couples insulin secretion to blood glucose levels, and maintains electrolyte balance.
The source paper by Fernandes et al. was published open access in Science Advances allowing readers to see the beautiful images of this channel with its four-part structure and selectivity filter. They claim it is the “first structure” published of Kir2.1. The average resolution is at 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.
This is the first time that the entire human Kir2.1 channel has been 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 a voltage between inner and outer membranes? And how do they know when to act?
The inward-rectification mechanism results from a block on the cytoplasmic side of the channels by endogenous polyamines and Mg2+ that plug the channel pore at depolarized potentials, resulting in decreased outward currents. The blockers are then removed from the pore when the K+ ions flow into the cell at hyperpolarized potentials. This voltage-dependent block results in efficient conduction of current only in the inward direction. In addition to being inwardly rectifying, Kir channels respond to a variety of intracellular messengers that directly control the 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 in 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 movies of the machine undergoing its precise conformational changes.
As the channel flexes, specific contacts between amino acids are made and broken to permit the accurate passage of potassium ions through the selectivity filter and three other constriction points, one called the G-loop where final potassium gating is thought to occur. The constrictions, as narrow as 1/5 of an Angstrom, act like gates that block everything until the right 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 the G-loop opening are most likely controlled by PIP2 binding. The replacement of R312 with histidine leads to a complete loss of the interaction network described above. Therefore, the interaction network integrity between subunits seems necessary for the proper allosteric transmission of the signal between R312 and the G-loop of the adjacent subunit upon PIP2 binding, which possibly allows the release of the constriction point on the G-loop. We can then hypothesize a PIP2-dependent G-loop gating mechanism that consists of the following: PIP2 binding triggers local conformational changes in the position of the side and main chains of R218 and K219, which, because of the structural proximity, lead to significant changes in the position of H221, displacing it laterally toward the intracellular medium. This movement would, in turn, cause E303 and R312 of the adjacent chain to move in the same direction, causing the G-loop to open.
Without meaning to overdo the technical jargon, the design only becomes evident in the details. Once again, readers will look in vain for any mention of how this channel emerged or evolved.