The Limiting Effect of Cytoplasmic Volume and Microtubule Dynamics

Mitotic spindles play a key role in cellular division. These structures, which are composed of dynamic filaments called microtubules, are responsible for separation and segregation of chromosomes during mitosis. Spindles must be the correct shape and size to insure fidelity of this process, however, their formation and assembly are still not entirely understood. For example, the mechanisms that govern spindle shape and determine individual spindle size for a given cell type are still unknown. Based on evidence from recent studies of spindle scaling, in which spindle size effectively scaled with cell size, we hypothesize that within small cytoplasmic volumes, spindle building blocks become limiting and thereby limit spindle size. This hypothesis predicts that microtubule dynamics within the spindle will be adversely affected by changes in cytoplasmic volume. By combining cell-free cytoplasmic extracts, microfluidics, and confocal microscopy, we hope to measure changes in microtubule dynamics to elucidate the relationship between cell volume and spindle size within the cell. Background: Cellular division is a topic most individuals can conceptualize. For a cell to faithfully replicate and divide, it must ensure that the new daughter cells are given equal amounts of the same genetic material that was produced during DNA replication. Mitotic spindles play a key role in cellular division. They are responsible for the segregation of chromosomes into daughter cells during mitosis. These spindles are composed of microtubules, filamentous polymers made up of heterodimeric protein subunits called tubulin (Feit et al, 1971). Microtubules are inherently dynamic filaments in the sense that they must grow and shorten to function, e.g. to bind to and separate the chromosomes during mitosis. The dynamics of microtubules are determined by several different parameters including growth rate, shrinkage rate, and the frequencies of transitions between the two states (Akhmanova and Steinmetz, 2015). It makes sense that these dynamics might affect spindle size. Indeed, the rate at which microtubules grow, the tubulin mass that has been incorporated into the microtubules, and the average length of the microtubules all affect spindle size. For example, within Xenopus laevis, a balance of mass is required between the rate of tubulin entering spindle and that of tubulin leaving spindle assembly (Reber et al, 2013). In order to maintain homeostasis, every living cell must be able to regulate the size of its internal parts regardless of its own size. The geometric relationship is termed scaling and comes in two forms: isometric scaling and allometric scaling. When two objects that are geometrically similar, for example a small equilateral triangle and a larger equilateral triangle, they are said to exhibit isometry. Isometric scaling would mean that the relationships between volume and surface area would remain constant between different sizes of cells because the cells are geometrically similar. Allometry refers to a change in shape in response to a change in size. This means that allometric scaling would result in not only a change in size, but shape. Scaling occurs at the extreme of cell sizes bacteria as small as 1μm in diameter and X. laevis blastomeres, which can be up to 1.2mm in diameter (some 1000 times larger) both must scale their replication machinery as a means for survival. In eukaryotes, failure of chromosome separation can lead to aneuploidy, which can lead to problems like Down’s Syndrome and cancer (Fang and Zhang, 2011). This scaling challenge is essential for normal development. Spindles must be in the correct size and shape in order effectively ensure fidelity of this process. Dynamics of microtubules affect spindle size and shape (Dumont and Mitchison, 2009). It is generally observed that, up until a point, as cell diameter increases, so does spindle length (Wühr et al, 2008). This is true up until a certain point where relationship plateaus (Wühr et al, 2008). This suggests that to the relationship between cell size and spindle size could be regulated by limiting components, changes in cytoplasmic composition that accompany developmental progression, or negative feedback induced by sterically constraining spindles in small volumes. It is known that enzymes are involved in the regulation of spindle size however this process is not entirely understood (Reber et al, 2013; Petry, 2016). Currently, a “limiting component” model is generally accepted (Good et al 2013, Hazel et al 2013). This mode posits that for a system of microtubules (e.g. a spindle) is built in small cellular environment, the cytoplasmic concentrations of spindle components and enzymes required to assemble them will be more greatly affected than in a larger cell building the same structure. For example, the enzyme XMAP215, which is part of a family of proteins that promotes microtubule growth, can directly catalyze the addition up to 25 tubulin dimers to the growing end of the molecule (Brouhard et al, 2008). This leads to the general question of not only how does cell size determine spindle size, but also the rate at which spindles are formed. We hypothesized that if spindle assembly occurs in a larger cell volume, then these spindles will be formed at a faster rate than cells of a smaller volume because they would be able to maintain a saturating number of enzyme molecules per growing microtubule end. In a small cellular volume, the relative starting concentration of that enzyme might still be the same, however the number of enzyme molecules per microtubule growing end would be sub-saturating, resulting in a slower rate of microtubule assembly. Cells of different volumes would still have the same concentrations of this enzyme. However, once microtubules begin to assemble, and the enzymes that bind them are effectively pulled out of solution to add tubulin subunits to growing ends, lowering the degree of saturation of this enzyme on those growing ends (Reber et al, 2013; Brouhard et al, 2008). X. laevis egg extracts have a long and extensive history for use as model cell-free systems. In 1985, Lohka and Maller demonstrated that nuclear envelope breakdown, chromosome condensation, and spindle assembly could all be studied iv vitro using extracts of amphibian eggs (Lohka and Maller, 1985). This system has been used to research several fundamental biological processes ranging from analyzing the effect cigarette smoke has on the inhibition of CFTR expression, to studying stability of the DNA replication fork (Moran et al, 2014; Hashimoto and Costanzo, 2011). X. laevis frogs were injected with hormones so they could lay eggs (as described in Desai et al, 1999). Once the eggs were harvested, they were washed with buffer and de-jellied, and packed into a test tube to remove any excess buffer. A crushing spin produced a stratified and fractionated extract from which the cytoplasmic layer was collected. This cytoplasmic extract is what we used to visualize and analyze microtubule growth. There are several advantages to using this cell-free system. It allows for easy controlling of in vitro processes, with protein biosynthesis being especially important for this experiment. Additionally, the absence of a cellular membrane usually allows for a faster reaction then microbial systems with cellular membranes (Zhu et al, 2013; Martín del Campo et al, 2013). Droplet microfluidics allow us to perform laboratory operations on very small scales (Mashaghi, et al, 2016). We could take our cytoplasmic extract and emulsify it with a surfactant and oil phase. As the name implies, this is done at a microscopic scale, at a size which we can control. Filling a single device can provide thousands of individual extract droplets. This provides us with flexibility to search under the microscope for the droplets with specific conditions we are interested in