Pavel Kraikivski

CLIP-170-Dependent Capture of Membrane Organelles by Microtubules Initiates Minus-End Directed Transport

Cytoplasmic microtubules (MTs) continuously grow and shorten at free plus ends. During mitosis, this dynamic behavior allows MTs to capture chromosomes to initiate their movement to the spindle poles; however, the role of MT dynamics in capturing organelles for transport in interphase cells has not been demonstrated. Here we use Xenopus melanophores to test the hypothesis that MT dynamics significantly contribute to the efficiency of MT minus-end directed transport of membrane organelles. We demonstrate that initiation of transport of membrane-bounded melanosomes (pigment granules) to the cell center involves their capture by MT plus ends, and that inhibition of MT dynamics or loss of the MT plus-end tracking protein CLIP-170 from MT tips dramatically inhibits pigment aggregation. We conclude that MT dynamics are required for the initiation of MT transport of membrane organelles in interphase cells, and that +TIPs such as CLIP-170 play an important role in this process.

Diffusion in Cytoplasm: Effects of Excluded Volume Due to Internal Membranes and Cytoskeletal Structures

The intricate geometry of cytoskeletal networks and internal membranes causes the space available for diffusion in cytoplasm to be convoluted, thereby affecting macromolecule diffusivity. We present a first systematic computational study of this effect by approximating intracellular structures as mixtures of random overlapping obstacles of various shapes. Effective diffusion coefficients are computed using a fast homogenization technique. It is found that a simple two-parameter power law provides a remarkably accurate description of effective diffusion over the entire range of volume fractions and for any given composition of structures. This universality allows for fast computation of diffusion coefficients, once the obstacle shapes and volume fractions are specified. We demonstrate that the excluded volume effect alone can account for a four-to-sixfold reduction in diffusive transport in cells, relative to diffusion in vitro. The study lays the foundation for an accurate coarse-grain formulation that would account for cytoplasm heterogeneity on a micron scale and binding of tracers to intracellular structures.

From START to FINISH: computational analysis of cell cycle control in budding yeast

In the cell division cycle of budding yeast, START refers to a set of tightly linked events that prepare a cell for budding and DNA replication, and FINISH denotes the interrelated events by which the cell exits from mitosis and divides into mother and daughter cells. On the basis of recent progress made by molecular biologists in characterizing the genes and proteins that control START and FINISH, we crafted a new mathematical model of cell cycle progression in yeast. Our model exploits a natural separation of time scales in the cell cycle control network to construct a system of differential-algebraic equations for protein synthesis and degradation, post-translational modifications, and rapid formation and dissociation of multimeric complexes. The model provides a unified account of the observed phenotypes of 257 mutant yeast strains (98% of the 263 strains in the data set used to constrain the model). We then use the model to predict the phenotypes of 30 novel combinations of mutant alleles. Our comprehensive model of the molecular events controlling cell cycle progression in budding yeast has both explanatory and predictive power. Future experimental tests of the model’s predictions will be useful to refine the underlying molecular mechanism, to constrain the adjustable parameters of the model, and to provide new insights into how the cell division cycle is regulated in budding yeast.

Barrier crossing of semiflexible polymers

We consider the motion of semiflexible polymers in double-well potentials. We calculate shape, energy, and effective diffusion constant of kink excitations, and in particular their dependence on the bending rigidity of the semiflexible polymer. For symmetric potentials, the kink motion is purely diffusive, whereas kink motion becomes directed in the presence of a driving force on the polymer. We determine the average velocity of the semiflexible polymer based on the kink dynamics. The Kramers escape over the potential barriers proceeds by nucleation and diffusive motion of kink-antikink pairs, the relaxation to the straight configuration by annihilation of kink-antikink pairs. Our results apply to the activated motion of biopolymers such as DNA and actin filaments or synthetic polyelectrolytes on structured substrates.

Regulation of microtubule-based transport by MAP4

Binding to microtubules of Xenopus microtubule-associated protein 4 (XMAP4) negatively regulates dynein-dependent movement of membrane organelles and positively regulates kinesin-2–based movement. Phosphorylation reduces binding of XMAP4 to microtubules and therefore regulates the direction of microtubule-based transport.

Implementing both short- and long-working-distance optical trappings into a commercial microscope

Optical tweezers are now a widespread tool based on three-dimensional trapping by a tightly focused single laser beam. This configuration only works with large numerical aperture and short-working-distance (SWD) objectives, restricting optical manipulation to the high magnification end of the microscope nosepiece. Certain applications of optical trapping demand long-working distances (LWDs) at moderate magnification, imposing a more complex two-beam trapping configuration. In this article, we describe a complete setup that incorporates both SWD and LWD optical trapping functionalities into a single Axiovert 200M Zeiss microscope. We evaluate the performance of the setup in both trapping modes with latex particles, either fluorescent or not, of different sizes, in the 1–20μm range. We provide practical information allowing for optimal configuration of the two-beam geometry, in relation with longitudinal and lateral stabilities of the trap.

Stimulation of the CLIP-170--dependent capture of membrane organelles by microtubules through fine tuning of microtubule assembly dynamics.

A combination of experimental and computational approaches shows that signals that stimulate minus end–directed transport of membrane organelles along microtubules change microtubule assembly dynamics in a way that enhances organelle capture by the growing microtubule tips.

Engineered Tug-of-War Between Kinesin and Dynein Controls Direction of Microtubule Based Transport In Vivo.

Bidirectional transport of membrane organelles along microtubules (MTs) is driven by plus‐end directed kinesins and minus‐end directed dynein bound to the same cargo. Activities of opposing MT motors produce bidirectional movement of membrane organelles and cytoplasmic particles along MT transport tracks. Directionality of MT‐based transport might be controlled by a protein complex that determines which motor type is active at any given moment of time, or determined by the outcome of a tug‐of‐war between MT motors dragging cargo organelles in opposite directions. However, evidence in support of each mechanisms of regulation is based mostly on the results of theoretical analyses or indirect experimental data. Here, we test whether the direction of movement of membrane organelles in vivo can be controlled by the tug‐of‐war between opposing MT motors alone, by attaching a large number of kinesin‐1 motors to organelles transported by dynein to minus‐ends of MTs. We find that recruitment of kinesin significantly reduces the length and velocity of minus‐end‐directed dynein‐dependent MT runs, leading to a reversal of the overall direction of dynein‐driven organelles in vivo. Therefore, in the absence of external regulators tug‐of‐war between opposing MT motors alone is sufficient to determine the directionality of MT transport in vivo.

CrossPlan: systematic planning of genetic crosses to validate mathematical models

Motivation Mathematical models of cellular processes can systematically predict the phenotypes of novel combinations of multi‐gene mutations. Searching for informative predictions and prioritizing them for experimental validation is challenging since the number of possible combinations grows exponentially in the number of mutations. Moreover, keeping track of the crosses needed to make new mutants and planning sequences of experiments is unmanageable when the experimenter is deluged by hundreds of potentially informative predictions to test. Results We present CrossPlan, a novel methodology for systematically planning genetic crosses to make a set of target mutants from a set of source mutants. We base our approach on a generic experimental workflow used in performing genetic crosses in budding yeast. We prove that the CrossPlan problem is NP‐complete. We develop an integer‐linear‐program (ILP) to maximize the number of target mutants that we can make under certain experimental constraints. We apply our method to a comprehensive mathematical model of the protein regulatory network controlling cell division in budding yeast. We also extend our solution to incorporate other experimental conditions such as a delay factor that decides the availability of a mutant and genetic markers to confirm gene deletions. The experimental flow that underlies our work is quite generic and our ILP‐based algorithm is easy to modify. Hence, our framework should be relevant in plant and animal systems as well. Availability and implementation CrossPlan code is freely available under GNU General Public Licence v3.0 at https://github.com/Murali‐group/crossplan

Quantifying a Pathway: Kinetic Analysis of Actin Dendritic Nucleation

Progress in uncovering the reaction networks that underlie important cell functions is laying the groundwork for quantitative identification of protein-interaction pathways. Since direct measurement of rate constants is not always feasible, the parameters are often inferred from multiple pieces of data using kinetic analyses based on appropriate mathematical models. The success of this approach relies on the sufficiency of available experimental data for a unique parameterization of the network. The concept of a rate-limiting step is applied to the analysis of experimental data that are usually used to quantify a pathway of actin dendritic nucleation, the Arp2/3-mediated mechanism that enables rapid changes of cell shape in response to external cues. The method yields analytical descriptions of the dynamics of polymerized actin and provides insights into how the experimental curves should be analyzed. It is shown that dynamics measured by pyrene-labeled actin assays with varying Arp2/3 concentrations are equally well described by two different rate-limiting steps: 1), binding of a nucleating complex to the side of a preexisting filament; or 2), its subsequent activation. To distinguish between the alternatives, we propose experiments with varying concentrations of actin monomers, taking advantage of the fact that the number of branches in the two cases depends differently on the initial monomer concentration. The idea is tested by simulating the proposed experiments with the use of spatial stochastic modeling.