Analysis of biological noise in the flagellar length control system.

Any proposed mechanism for organelle size control should be able to account not only for average size but also for the variation in size. We analyzed cell-to-cell variation and within-cell variation of length for the two flagella in Chlamydomonas, finding that cell-to-cell variation is dominated by cell size, whereas within-cell variation results from dynamic fluctuations. Fluctuation analysis suggests tubulin assembly is not directly coupled with intraflagellar transport (IFT) and that the observed length fluctuations reflect tubulin assembly and disassembly events involving large numbers of tubulin dimers. Length variation is increased in long-flagella mutants, an effect consistent with theoretical models for flagellar length regulation. Cells with unequal flagellar lengths show impaired swimming but improved gliding, raising the possibility that cells have evolved mechanisms to tune biological noise in flagellar length. Analysis of noise at the level of organelle size provides a way to probe the mechanisms determining cell geometry.

Speed and Diffusion of Kinesin-2 Are Competing Limiting Factors in Flagellar Length-Control Model.

Flagellar length control in Chlamydomonas is a tractable model system for studying the general question of organelle size regulation. We have previously proposed that the diffusive return of the kinesin motor that powers intraflagellar transport can play a key role in length regulation. Here, we explore how the motor speed and diffusion coefficient for the return of kinesin-2 affect flagellar growth kinetics. We find that the system can exist in two distinct regimes, one dominated by motor speed and one by diffusion coefficient. Depending on length, a flagellum can switch between these regimes. Our results indicate that mutations can affect the length in distinct ways. We discuss our theory's implication for flagellar growth influenced by beating and provide possible explanations for the experimental observation that a beating flagellum is usually longer than its immotile mutant. These results demonstrate how our simple model can suggest explanations for mutant phenotypes.

Extending chemical perturbations of the ubiquitin fitness landscape in a classroom setting reveals new constraints on sequence tolerance.

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary timescales. Building on our previous work (Mavor et al., 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl2, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-Fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.

Diffusion as a Ruler: Modeling Kinesin Diffusion as a Length Sensor for Intraflagellar Transport.

An important question in cell biology is whether cells are able to measure size, either whole cell size or organelle size. Perhaps cells have an internal chemical representation of size that can be used to precisely regulate growth, or perhaps size is just an accident that emerges due to constraint of nutrients. The eukaryotic flagellum is an ideal model for studying size sensing and control because its linear geometry makes it essentially one-dimensional, greatly simplifying mathematical modeling. The assembly of flagella is regulated by intraflagellar transport (IFT), in which kinesin motors carry cargo adaptors for flagellar proteins along the flagellum and then deposit them at the tip, lengthening the flagellum. The rate at which IFT motors are recruited to begin transport into the flagellum is anticorrelated with the flagellar length, implying some kind of communication between the base and the tip and possibly indicating that cells contain some mechanism for measuring flagellar length. Although it is possible to imagine many complex scenarios in which additional signaling molecules sense length and carry feedback signals to the cell body to control IFT, might the already-known components of the IFT system be sufficient to allow length dependence of IFT? Here we investigate a model in which the anterograde kinesin motors unbind after cargo delivery, diffuse back to the base, and are subsequently reused to power entry of new IFT trains into the flagellum. By mathematically modeling and simulating such a system, we are able to show that the diffusion time of the motors can in principle be sufficient to serve as a proxy for length measurement. We found that the diffusion model can not only achieve a stable steady-state length without the addition of any other signaling molecules or pathways, but also is able to produce the anticorrelation between length and IFT recruitment rate that has been observed in quantitative imaging studies.

Cytoplasmic dynein transports cargos via load-sharing between the heads.

Cytoplasmic dynein is a motor protein that walks along microtubules (MTs) and performs mechanical work to power a variety of cellular processes. It remains unclear how a dynein dimer is able to transport cargos against load without coordinating the stepping cycles of its two heads. Here by using a DNA-tethered optical trapping geometry, we find that the force-generating step of a head occurs in the MT-bound state, while the 'primed' unbound state is highly diffusional and only weakly biased to step towards the MT-minus end. The stall forces of the individual heads are additive, with both heads contributing equally to the maximal force production of the dimer. On the basis of these results, we propose that the heads of dynein utilize a 'load-sharing' mechanism, unlike kinesin and myosin. This mechanism may allow dynein to work against hindering forces larger than the maximal force produced by a single head.