The final stages of the fundamental process of cell division are driven, in most cases, by a contractile actomyosin ring, but little is known about how this ring gets assembled or performs its role. Science Life spoke with post-doctoral fellow Dennis Zimmermann, PhD – in the laboratory of David Kovar, PhD, Professor of Molecular Genetics and Cell Biology, and of Biochemistry and Molecular Biology. They were the first and senior authors of a study published September 26, 2016, in Nature Communications.
Their project, “Mechanoregulated inhibition of formin facilitates contractile actomyosin ring assembly,” describes the underlying molecular principles that govern the highly coordinated assembly of the contractile actomyosin ring during cell division. Zimmerman is now an independent research fellow at the Massachusetts Institute of Technology’s Koch Institute for Integrative Cancer Research.
What is a contractile actomyosin ring?
The contractile actomyosin ring is a belt-like structure that (most) dividing cells assemble at the cell equator after chromosome separation during the final stage of cell division. The contractile ring plays a crucial role in most dividing animal cells. It constricts the mother cell, thereby mediating the physical separation into two daughter cells.
What is actomyosin, and what is formin?
Actomyosin describes filamentous actin network structures that interact with myosin motor proteins. This provides these networks with the ability to produce forces powerful enough to work against the internal turgor pressure of a dividing cell. The contractile ring is only one of many different actomyosin networks. The classical example is a muscle sarcomere where myosin motors and actin make up the core contractile unit of a muscle fiber.
Formins describe a conserved family of actin-binding proteins that have evolved to drive the continuous assembly of long straight actin filaments.
Why and how do you study this in yeast?
The actin cytoskeleton is a versatile, complex machinery that encompasses many different components. Each one carries out its own specific function at the right time and place. Human cells can assemble more than 20 different actin networks, employing dozens of different actin-binding proteins. The formin family in humans, for example, comprises more than 15 members.
In contrast, the unicellular fission yeast (Schizosaccharomyces pombe) has been serving as a popular model organism because it assembles only three main actin networks, while encoding only three different formins. Working with this reduced set of cytoskeletal actin network components, we are able to manipulate and visualize specific sets of actin-binding proteins (e.g. formins) with relative ease.
The parts list of actin-binding proteins involved in building the fission yeast contractile ring have been identified in previous studies. It is still unclear, however, how the different factors collaborate and regulate each other at the molecular level in order to facilitate proper ring assembly.
Therefore, we set out to reconstitute in vitro (i.e. to rebuild outside the cell) the previously proposed model of contractile ring assembly using purified and fluorescently labeled versions of the essential ring components: actin, formin and myosin. Using specialized multi-color microscopy (a total internal reflection fluorescence microscope), we were able to study the behavior of individual formin molecules that are bound to an elongating actin filament that at the same time experiences pulling forces by engaging myosin motors. We also used confocal fluorescence microscopy of live fission yeast cells containing fluorescently labeled versions of the same proteins to validate the relevance of our in vitro findings in vivo.
Can you explain – at a very basic level – how this process works?
In this study, we demonstrate the first minimal component reconstitution of the Search-Capture-Pull model for contractile ring assembly.
In this model, formins, anchored to ring precursor structures at the cell membrane, assemble ‘searching’ actin filaments using the cytoplasmic pool of actin filament building blocks, while remaining continuously associated with the elongating actin filament. Class II myosin motors then ‘capture’ the ‘searching’ filament and ‘pull’ on the filament, thereby bringing neighbouring ring precursor structures closer together.
This behaviour has never been recapitulated in vitro, so the underlying molecular mechanisms and ensemble properties of components facilitating node condensation were unknown.
We discovered that the application of minute (sub-piconewton) forces by the physiological force generator myosin Myo2 to formin Cdc12-bound actin filaments results in the reversible mechano-inhibition of Cdc12’s ability to polymerize actin filaments. Mechanistically, we identified the formin domain that relays the force-sensitive response. Quantitative modelling suggests that those small forces may suffice to stretch the force-sensitive domain, which in turn impedes formin-mediated actin filament elongation. Finally, live cell imaging of mechano-insensitive formin mutant cells established that mechano-inhibition of formin Cdc12 is required to effectively condense contractile ring precursors, thereby enabling efficient cytokinesis in vivo.
What parts of the process can go awry? What damage can that do?
One of the most critical points during cell division is ensuring proper chromosome segregation. This step precedes ring formation and is tightly regulated. Cells do not enter the final phase of cell division, during which ring assembly occurs, until chromosome separation has occurred. This is a crucial step in obtaining two healthy progenitor cells. Improper ring constriction can cause chromosome mis-segregation, which leads to dysfunction or cell death.
How does the cell prevent that?
The different ways in which cells prevent improper ring assembly have been under extensive investigation for years. Some questions have been answered; even more have arisen. One crucial part of proper ring assembly and successful cell division is the correct placement of the so-called cleavage site, the point at which the actual contractile ring will assemble and constrict.
This process is largely determined by signaling pathways that drive the initial localization and activation of various ring components. What is less well understood is how components like myosin and formin are regulated once activated. This is what motivated us to study the underlying molecular mechanisms acting during the assembly of the contractile actomyosin ring.
Formin’s role involves mechanical force rather than chemical signaling. How do these processes co-exist?
I think chemical signaling and the influence of mechanical force almost always co-exist. Therefore, the question is not so much how these two kinds of modes of regulation co-exist, but how do they impact and counter-balance each other. This opens up a whole new and exciting layer of regulation that requires further investigation.
Here’s one way to think of it. Chemical signaling cues often initiate (or terminate) specific responses. They set the stage for a specific process that’s about to happen. Mechanical forces, on the other hand, provide the cell with an adjustable dial-like tool through which a given response can be tuned to accommodate ever-changing cellular constraints.
How significant, as a rule, is mechanical as compared to chemical force?
They are co-dependent. Both signaling-mediated and mechano-regulated forces must co-exist to ensure the functionality of a given cellular process under physiological conditions.
Which other important cellular processes rely on mechanotransduction?
Quite a few. This is widespread. Examples would include cell adhesion, motility and migration, tissue development, wound healing. This area of biological inquiry will keep us busy for a long time.
The United States Department of Defense, the German Research Foundation and the National Institutes of Health supported the research. Additional authors include Kaitlin Homa, Glen Hocky and Gregory Voth from the University of Chicago; Luther Pollard and Kathleen Trybus from the University of Vermont; and Enrique De La Cruz from Yale University.