On the Frontline: Allen Liu—the Mechanome and Synthetic Cell Development

Allen Liu - 2
ALLEN P. LIU

I spoke last week with mechanical and biomedical engineer Allen Liu, who’s now on sabbatical at Max Planck Institute of Biochemistry in Martinsried just outside Munich—one of Europe’s research centers on synthetic cell development.  His own lab is at the University of Michigan, so he called me from Germany for our scheduled interview.  We were first in touch regarding my coverage of the Dutch Synthetic Cell Symposium at Delft where he was a keynote speaker this past summer. 

Allen Liu is originally from Taiwan and his cultural roots are clearly evident in his gracious professional communications. Not surprisingly, he’s received a National Science Foundation grant for his synthetic (artificial) cell project and is currently in the swirl of some of Europe’s most cutting edge science.  Liu was also one of the speakers—and perhaps the youngest—at the NSF meeting in Alexandria, Virginia on synthetic and artificial cell development earlier this year.

Allen Liu heads the Liu Lab at the University of Michigan—where the focus is on the mechanobiology of biological membranes—and he is a professor in the university’s departments of mechanical and biomedical engineering. 

Liu also serves as associate editor of RSC Advances and is an advisory editorial board member of WIREs Nanomedicine and Nanobiotechnology as well as an editorial board member of Heliyon.

Awards include:  Emerging Investigator (Chemical Communications, 2017); Rising Star Award (BMES-Cellular and Molecular Bioengineering, 2017); Young Innovator in Cellular and Molecular Bioengineering (2014); NIH Director’s New Innovator Award (2012), among others. 

Allen Liu’s BSc is in biochemistry, with honors, from the University of British Columbia and his PhD from the University of California, Berkeley (working with Daniel Fletcher). He was a postdoctoral fellow in cell biology at Scripps Research Institute (working with Gaudenz Danuser and Sandra Schmid).

Our interview follows.

Suzan Mazur: I detect a faint Canadian accent in your NSF video presentation on the artificial cell. Are you a native of Canada?

Allen Liu:  I was born in Taiwan actually and moved to Canada as a teenager. I lived in Canada for eight years and did my undergrad studies there. I’ve been living and working in the US since then, except for my current sabbatical here in Germany at the Max Planck Institute of Biochemistry.  I did my PhD, my postdoc and started my lab all in the US.

Suzan Mazur: Do you come from a science family?

Allen Liu: My dad has a Master’s degree in chemical engineering. Mom specialized in pharmacy in medical school.  Both of my parents trained in Taiwan.  So, as a kid I had a keen interest in science.

Suzan Mazur:  Do you have time for other interests outside the lab?  Hobbies?

Allen Liu: I find that I have less and less free time outside the lab. Work as a scientist is pretty intense and I do work a lot.  But I like sports, particularly tennis. And I very much enjoy spending time with my family. I have two daughters now.

Suzan Mazur:  Is your wife a scientist?

Allen Liu:  My wife trained as a biologist and educator.  Before we had our daughters she taught at a Montessori school—kids age zero to six.  She is now taking care of our two young daughters. 

Suzan Mazur: You seem to be the one scientist who has presented at all the pivotal meetings in the last six months:  (1) the National Science Foundation workshop on synthetic and artificial cells, (2) the Dutch Synthetic Cell Symposium, (3) “The Mechanome in Action” meeting.  You also attended the Munich “Molecular Origins of Life” conference in October. Let’s start with what you understand by the term “mechanome.”

Allen Liu:  Mechanome refers to the set of proteins or molecular entities that sense or respond to forces.  How cells respond to mechanical cues, etc.

Suzan Mazur:  A decade ago, then-MIT bioengineer Matthew Lang writing in the National Academy of Engineering journal Expanding Frontiers of Engineering talked about sequencing the mechanome, i.e., “the role of force, mechanics and machinery in biology,” and expressed the following:

“The design of biological motors can be classified by cataloging the motor’s general structural features, fuel type, stepping distance, stall force, and other mechanical parameters. Detailed measurements of the motility cycles and underlying mechanisms for motility also provide information about how these mechanisms work.  Ultimately ‘sequencing’ the mechanome will lead to the discovery of the design features of biological motors in general, enabling us to catalogue them and outline the rules that govern their behavior.”

Lang concluded:

“Once we understand the physical rules that govern biological systems and can measure nature’s machinery, we should eventually be able to ‘sequence’ the mechanome.”

My question is— how far along are we in sequencing the mechanome?

Allen Liu:  I don’t see it as something that can be ‘sequenced’, as we do with the genome. I think Matt had an important insight that if we measure the forces and displacements of proteins under forces, we may be able to decipher the design principle of molecular machines. If you think about all the proteins that sense or respond to forces, one can argue that in principle all proteins are sensitive to forces. This is because proteins are folded into three-dimensional structures by non-covalent interactions.  And if you apply a force by grabbing onto a single molecule and extend it, the protein will unfold. So by their nature, all proteins are sensitive to forces. 

The key thing the field is trying to understand now is that if a force is applied, how does it change the energy landscape of the molecular interactions. How does that force facilitate binding or unbinding to other molecular entities? We still don’t know which proteins have cryptic sites that open upon physiological force applications. To a certain extent, we have not identified all the key molecular players in force transduction. 

We learned only about eight years ago [in 2010], for example, of the existence of mammalian ion channels that are mechanically activated and are involved in touch and pain sensation.  They are called Piezo1 and Piezo2.  

[piezo, from the Greek word, piesi—meaning pressure] 

figure cell + legend

Suzan Mazur:  That’s quite interesting.  I didn’t know about Piezo1 and 2. 

Can you separate the mechanome from the genome?

Allen Liu:  I see the mechanome in a way as a subset of the proteome—sensitive mechanical forces as a subset.  In describing the mechanome, it’s not an individual protein that does the job.  A set of proteins is required to orchestrate mechanotransduction

One example is force applied to focal adhesion.  When you stretch the N-terminal region of talin, it reveals cryptic binding site to vinculin.  For the protein to be sensitive to mechanical force, the action is often orchestrated not by one single protein but by a set of proteins.

Then, of course, there’s the cytoskeleton, which is involved in contractility.  A sort of chain reaction happens—cells exert force on the substrate and neighboring cells as part of the mechanotransduction process.

Suzan Mazur:  Eberhard Bodenschatz said—at that same NSF meeting in May where you presented—that scientists at Max Planck are trying to see how far they can go in developing a synthetic cell WITHOUT introducing a genome.  Unlike the Dutch who have added a genome to the equation. What are your thoughts?

Allen Liu:  I do remember Eberhard making that statement.  What I gather from that is, it depends on what you want to do with a synthetic cell.  It may not need to divide.  It can be an entity that can perform certain functions. To do that you can reconstitute everything from the bottom-up, by including purified proteins and encapsulate all of that in a lipid bilayer vesicle. That is essentially what the PIs [Principal Investigators] in the MaxSynBio groups are doing, although the MaxSynBio groups, to my knowledge, are targeting multiple areas (energy supply, metabolism, growth, division, etc.) as well as developing platform technology (microfluidics and cell-free expression).

Suzan Mazur:  You’re now doing research in one of these groups, in Petra Schwille’s lab. So, would you say the MaxSynBio groups are making an artificial or synthethic cell?

Allen Liu:  An artificial cell that does not have any encoded components.  It can still come from purifying a protein out of a host cell like E. coli.  A major approach is based on protein reconstitution.

The way that this could work is—if you think about the cells in our bodies that do not have a nucleus, like red blood cells and platelets.  Although these cells do not have a nucleus, they perform very sophisticated functions.  For instance, a platelet is just ~2-3 microns in size, yet it has all the protein machinery enclosed within its own cell membrane and can function in blood coagulation without having any genetically encoded components.

Petra’s lab encompasses several areas, but with the major focus on dynamic pattern formation of cell division machineries.

Suzan Mazur:  Can you tell me about the patent you hold regarding an artificial cell?

Allen Liu:  That was part of my PhD work with Dan Fletcher at Berkeley.  The basic idea is to use a vesicle and purified proteins as a platform for an artificial cell with controlled membrane compositions and asymmetry.  At the time we developed a strategy to encapsulate using a liquid jet.

Suzan Mazur: The Liu Lab at the University of Michigan is particularly interested in the mechanobiology of the cell lipid membrane.  Would you briefly describe your hypothesis about cell tension and membrane trafficking and the significance of this in building an artificial cell?

Allen Liu:  The hypothesis is currently more relevant to living cells.  At the moment it would be a bit difficult to extrapolate that for synthetic or artificial cells.

We are building mechanosensitive cells—artificial cells that will basically respond to mechanical forces.  In this case, we are mimicking the basic process that cells can sense and respond to forces.  This is work we’ve done on our own as well as in collaboration with Vincent Noireaux at the University of Minnesota

Basically, we are incorporating channel proteins that are known to respond to membrane tension in the bilayer. If we incorporate this successfully, the artificial cell senses elevated membrane tension and will open.  A pore, like a little door, enables molecules to go in and out.  We’re writing this paper up now.

Suzan Mazur:  Are you looking at neutron scattering for information in designing your artificial cell?  It’s fascinating what they can see with neutron scattering.  I’ve had recent conversations about this with John Katsaras who’s at Oak Ridge National Laboratory and Tommy Nylander at  Lund University/ ESS.  They’re both membrane specialists.

Allen Liu:  I am not working with neutron scattering, but I’m aware of some of the classic work that’s been done.  That you can see the structure of membranes precisely with neutron scattering.

Suzan Mazur:  And motion.

Allen Liu:  That’s right.  We’re mostly using fluorescence light microscopy as our main tool coupled with careful image analysis.

But just to finish my comment about membrane trafficking—this has very little to do with the synthetic cell at the moment.

In my lab I have also been very interested in understanding the dynamics of membranes from the perspective of plasma membrane in living cells.

As a postdoc I studied endocytosis.  This is a process where cells internalize plasma membrane into endocytic vesicles.  The classic paradigm that was set up before by Columbia University biologist Michael Sheetz [a founding director of Singapore’s Mechanobiology Institute] was that if you had a tense membrane, it becomes very difficult to endocytose. Because the cells like a membrane at a certain tension.

So it has to do with the homeostasis of the membrane. The tension of the membrane is at a relative set point, When the tension is high, you exocytose, and when the tension is low, you endocytose.   

To me this is relevant regarding communication with the external environment.  If you want to cross information in and out—you know the cell uses receptors in binding, uses light but also uses force.  So, if you just consider receptor-ligand binding in signal transduction pathways, one of the key players is the receptor.  The receptor can be internalized. 

There’s an intersection between mechanobiology and endocytosis.  This is an intersection that has not been looked at very much.  People I meet in the mechanobiology world focus on focal adhesion, cell-cell tension, and cytoskeletal tension, but rarely look at the effect on membrane trafficking.  It’s something my lab is actively working on right now.

Suzan Mazur:  Can you say what you’re working on there in Petra Schwille’s lab in Germany?

Allen Liu:  If you look at natural cells—there are compartments, there’s a nucleus, mitochondria, etc.  I’m looking at the engineering inside the cell.

I’m thinking about an artificial cell.  My perspective on the distinction between synthetic and artificial is this.  If I’m making something, say naturally extracted glucose, using a synthetic as opposed to a natural approach—then that is synthetic. If I’m making something that does not exist in nature—it’s artificial. 

But artificial cells are now also being referred to as synthetic cells. Funding agencies, like NSF, seem to have settled on the term synthetic cell to describe both artificial and synthetic cells.  I too switched to NSF’s preferred use of synthetic cell terminology in my recent project proposal that NSF funded.

Suzan Mazur:  Can you tell me a bit more about your current research?

Allen Liu:  One area that I’m happy to share involves liquid-liquid phase separation in organizing reactions within an artificial cell. 

A decade or so ago there was a big discovery in cell biology:  Compartments in cells that are not lipid bound—membrane-less organelles of liquid-nature that can fuse with one another, dissolve and grow.  This discovery inspired scientists doing synthetic cell research to investigate the phenomenon. It has now been looked at quite a bit.

Christine Keating, at Penn State, has actually been studying liquid-liquid phase separation for 15-20 years—primarily using polymers of dextran and PEG [polyethylene glycol].  But short polymers, coacervates can also be used.

I’m experimenting with these systems to see if there’s a way to form droplets within a cell-free expression system that can alter protein expression. The idea is to create an artificial cell with an internal organelle.  A sort of internal organelle that then specializes in one function.  I’m experimenting with liquid-liquid phase separation to see how this can be used in making artificial cells.

Suzan Mazur: Thank you. You raised several questions at the NSF meeting:  Can we build an artificial cell that couples mechanical input to a biochemical output?  Can we assemble biological components?  Can we insert membrane protein to build an artificial nucleus?  Are we closer to answering these questions six months later?

Allen Liu:  The first one and second one my lab has worked on extensively.  So I think we can say yes.  The third point about building an artificial nucleus—no, that has not been done.  I have just submitted a proposal on this but it has not been funded yet.

Suzan Mazur:  Were any of these origins of life and synthetic cell conferences in Europe video recorded?

Allen Liu:  No, I don’t think so.

Suzan Mazur:  It would be good if the Europeans publicly streamed these meetings. 

Allen Liu:  I’ll speak to Petra to see what she thinks about it.

Suzan Mazur:  Thank you, Allen.

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