RESUMEN

A fundamental question in biology is whether the presence of non-reacting macromolecules in the cytoplasm affects the rates and extents of reversible association reactions, a phenomenon often referred to as 'macromolecular crowding.' Under certain conditions, crowding has been proposed to dramatically alter the kinetics and thermodynamics of chemical reactions, making it difficult to quantitatively relate rates and extents of reactions measured in vitro to those occurring in vivo. In this work, we use Brownian dynamics simulation and Monte Carlo methods to (1) quantify the overall thermodynamic and kinetic effects of crowding by independently investigating each step of reversible bimolecular association (i.e. translational diffusion, steric specific binding, and dissociation), and (2) provide an explicit, quantitative investigation of how the degree of steric specificity of protein dimerization influences crowding-mediated effects on association and dissociation. We find that k on decreases by ∼2-fold for non-steric specific reactions, and increases by ∼3-fold for highly steric specific reactions. In addition, k off decreases by only ∼30%-60% in the presence of crowders, depending on the strength of the bond between the reactant pair, so that the equilibrium constant is increased by ∼4-fold, at most. These results suggest that crowding-mediated effects on globular protein dimerization reactions in the cytoplasm are modulated by the steric specificity of the reactants, and that reversible protein-protein association is relatively insensitive to the physical presence of crowders (i.e. steric repulsion effects in the cytoplasm) for crowders of similar size and shape to reactants over a range of volume fractions (0-0.3).

Asunto(s)

RESUMEN

Microtubule-associated proteins (MAPs) are proteins that reversibly bind to and regulate microtubule dynamics and functions in vivo. We examined the dynamics of binding of a MAP called ensconsin (E-MAP-115) to microtubules in vivo. We used 5xGFP-EMTB, a construct in which the microtubule-binding domain of ensconsin (EMTB) is fused to five copies of green fluorescent protein (GFP), as a reporter molecule amenable to the use of fluorescent speckle microscopy. Fluorescent speckle microscopy (FSM) sequences and kymograph analyses showed rapid dynamics of speckles comprised of 5xGFP-EMTB in untreated cells. By contrast, in detergent-lysed cytoskeletons, speckles were not dynamic. Since detergent-lysed cytoskeletons differ from living cells in that they lack both ATP and dynamic microtubules, we used azide treatment to substantially reduce the level of ATP in living cells and we used Taxol to halt microtubule dynamics. Both treatments slowed the dynamics of 5xGFP-EMTB speckles observed by FSM. We also used fluorescence recovery after photobleaching (FRAP) to quantify the half-time of binding and dissociation of the 5xGFP-EMTB chimera and to compare this half-time to that of the full-length MAP molecule. In untreated cells, the t(g) of either 5xGFP-EMTB or full-length GFP-ensconsin was similarly rapid (approximately 4 seconds), while in ATP-reduced and Taxol-treated cells, t(g) was increased to 210 seconds and 40 seconds, respectively. In detergent-extracted cells no recovery was seen. Consistent with the rapid dynamics of 5xGFP-EMTB measured with fluorescent speckle microscopy and FRAP, we estimated that the affinity of the MAP for microtubules is approximately 40 microM in untreated living cells, compared with approximately 1 microM in vitro. However, K(D,app) was not significantly changed in the presence of azide and was increased to 110 microM in the presence of Taxol. To test whether changes in the phosphorylation state of cellular proteins might be responsible for altering the dynamics of ensconsin binding, we used FSM to monitor staurosporine-treated cells. Staurosporine treatment substantially halted dynamics of 5xGFP-EMTB speckles along MTs. Our results show that ensconsin is highly dynamic in its association with microtubules, and its microtubule association can be altered by in vivo phosphorylation events.

Asunto(s)

RESUMEN

To perform their myriad functions, tissues use specific cell-cell interactions that depend on the spatial ordering of multiple cell types. Recapitulating this spatial order in vitro will facilitate our understanding of function and failure in native and engineered tissue. One approach to achieving such high placement precision is to use optical forces to deposit cells directly. Toward this end, recent work with optical forces has shown that a wide range of particulate materials can be guided and deposited on surfaces to form arbitrary spatial patterns. Here we report that, when we use the light from a near-infrared diode laser focused through a low numerical aperture lens, individual embryonic chick spinal cord cells can be guided through culture medium and deposited on a glass surface to form small clusters of cells. In addition, we found that the laser light could be coupled into hollow optical fibers and that the cells could be guided inside the fibers over millimeter distances. The demonstration of fiber-based guidance extends by 2 orders of magnitude the distance over which optical manipulation can be performed with living cells. Cells guided into the fiber remained viable, as evidenced by normal cell adhesion and neurite outgrowth after exposure to the laser light. The results indicate that this particle deposition process, which we call "laser-guided direct writing," can be used to construct patterned arrays of tens to hundreds of cells using arbitrary numbers of cell types placed at arbitrary positions with micrometer-scale precision.

Asunto(s)

RESUMEN

Microtubules in living cells frequently bend and occasionally break, suggesting that relatively strong forces act on them. Bending implies an increase in microtubule lattice energy, which could in turn affect the kinetics and thermodynamics of microtubule-associated processes such as breaking. Here we show that the rate of microtubule breaking in fibroblast cells increases approximately 40-fold as the elastic energy stored in curved microtubules increases to > approximately 1 kT/tubulin dimer. In addition, the length-normalized breaking rate is sufficiently large (2.3 breaks x mm(-1) x minute(-1)) to infer that breaking is likely a major mechanism by which noncentrosomal microtubules are generated. Together the results suggest a physiologically important, microtubule-based mechanism for mechanochemical information processing in the cell.

Asunto(s)

RESUMEN

Laser-induced optical forces can be used to guide and deposit 100 nm - 10 microm-diameter particles onto solid surfaces in a process we call 'laser-guided direct writing'. Nearly any particulate material, including both biological and electronic materials, can be manipulated and deposited on surfaces with micrometer accuracy. Potential applications include three-dimensional cell patterning for tissue engineering, hybrid biological-electronic-device construction, and biochip-array fabrication.

Asunto(s)

RESUMEN

During development neurons extend and retract cytoskeletal structures, chiefly microtubules and filopodia, to process informational cues from the extracellular environment and thereby guide growth cone migration toward an appropriate synaptic partner. This cytoskeleton-based exploration is achieved by stochastic switching, with microtubules and filopodia alternating between growing and shortening phases apparently at random. If stabilizing signals are not detected during the growth phase, then the structures switch to a shortening state, from which they can again return to a growth phase, and so forth. A useful means of characterizing these stochastic processes in a model-independent way is by autocorrelation and spectral analysis. Previously, we compared experiment to theory by performing Monte Carlo simulations and computing the autocorrelation function and power spectrum from the simulated dynamics, an approach that is computationally intensive and requires recalculation whenever model parameters are changed. Here we present analytical expressions for the autocorrelation function and power spectrum, which compactly characterize microtubule and filopodial dynamics based on the stochastic, two-state model. The model assumes that the phase times are of variable duration and gamma-distributed, consistent with experimental evidence for microtubules assembled in vitro from purified tubulin. The analytical expressions permit the precise quantitative characterization of changes in microtubule and filopodial searching behavior corresponding to changes in the shape of the gamma distribution.

Asunto(s)

RESUMEN

Microtubule assembly is a complex process with individual microtubules alternating stochastically between extended periods of assembly and disassembly, a phenomenon known as dynamic instability. Since the discovery of dynamic instability, molecular models of assembly have generally assumed that tubulin incorporation into the microtubule lattice is primarily reaction-limited. Recently this assumption has been challenged and the importance of diffusion in microtubule assembly dynamics asserted on the basis of scaling arguments, with tubulin gradients predicted to extend over length scales exceeding a cell diameter, approximately 50 microns. To assess whether individual microtubules in vivo assemble at diffusion-limited rates and to predict the theoretical upper limit on the assembly rate, a steady-state mean-field model for the concentration of tubulin about a growing microtubule tip was developed. Using published parameter values for microtubule assembly in vivo (growth rate = 7 microns/min, diffusivity = 6 x 10(-12) m2/s, tubulin concentration = 10 microM), the model predicted that the tubulin concentration at the microtubule tip was approximately 89% of the concentration far from the tip, indicating that microtubule self-assembly is not diffusion-limited. Furthermore, the gradients extended less than approximately 50 nm (the equivalent of about two microtubule diameters) from the microtubule tip, a distance much less than a cell diameter. In addition, a general relation was developed to predict the diffusion-limited assembly rate from the diffusivity and bulk tubulin concentration. Using this relation, it was estimated that the maximum theoretical assembly rate is approximately 65 microns/min, above which tubulin can no longer diffuse rapidly enough to support faster growth.

Asunto(s)

RESUMEN

To examine whether microtubule dynamic instability can be rapidly regulated during interphase, we used video-enhanced differential interference contrast (DIC) microscopy to observe individual microtubules at the periphery of living newt lung epithelial cells. Microtubules were observed before and after perfusion with either the phosphatase inhibitor okadaic acid or the kinase inhibitors staurosporine or olomoucine. Addition of these inhibitors caused rapid changes in dynamic instability. Thirty to sixty seconds after perfusion with 0.2-1 microM okadaic acid, a 1.5-fold increase in elongation velocity and small increases in catastrophe and rescue frequencies were observed. In contrast, treatment with 40-200 nM staurosporine decreased microtubule elongation and shortening velocities approximately 2-fold, and catastrophes were slightly more frequent. Olomoucine, at 100 microM, had similar effects. Transition dynamics were further examined by probabilistic analysis, which showed that microtubules become more likely to undergo catastrophe as they elongated and more likely to undergo rescue as they shortened, an effect previously called microtubule "memory." This memory effect for catastrophes was observed in untreated and okadaic acid-treated cells but was abolished by staurosporine or olomoucine. In contrast, the memory effect for rescue was unaffected by these treatments, suggesting that catastrophe and rescue proceed via distinct, multistep mechanisms. Overall, these results demonstrate that microtubule assembly regulators can be altered rapidly by inhibition of either kinases or phosphatases and suggest that, in the absence of inhibitors, these regulators exist in a dynamic equilibrium between phosphorylated and dephosphorylated states.

Asunto(s)

RESUMEN

The controlled extension of neurites is essential not only for nervous system development, but also for effective nerve regeneration after injury. This process is critically dependent on microtubule assembly since axons fail to elongate in the presence of drugs which disrupt normal assembly dynamics. For this reason, neurite outgrowth is potentially controllable by manipulation of the assembly state of the intracellular array of microtubules. Therefore, understanding how microtubule assembly dynamics and neurite outgrowth are coupled, in the absence of drugs, can lend valuable insight into the control and guidance of the outgrowth process. In the present study we characterized the stochastic dynamics of neurite outgrowth and its corresponding microtubule array, which advances concomitantly with the advance of the nerve growth cone, the highly motile structure at the terminus of the growing neurite, using reported fluorescent microscopic image sequences (Tanaka and Kirschner, 1991, J. Cell Biol. 115:345-363). Although previously modeled as an uncorrelated random walk, the stochastic advance of the growth cone was found to be anticorrelated over a time scale of approximately 4 min, meaning that growth cone advances tended to be followed by growth cone retractions approximately 4 min later. The observed anticorrelation most likely reflects the periodic stops and starts of neurite outgrowth that have been reported anecdotally. A strikingly similar pattern of anticorrelation was also identified in the advance of the growth cone's microtubule array. Cross-correlation analysis showed that growth cone dynamics tended to precede microtubule dynamics on a time scale of approximately 0-2 min, while microtubules tended to precede growth cone dynamics on a approximately 0-20-s time scale, indicating a close temporal coupling between microtubule and growth cone dynamics. Finally, the scaling of the mean-squared displacements with time for both the growth cone and microtubules suggested a fractional Brownian motion model which accounts for the observed anticorrelation of growth cone and microtubule advance. (c) 1996 John Wiley & Sons, Inc.

RESUMEN

Microtubules are cytoskeletal filaments whose self-assembly occurs by abrupt switching between states of roughly constant growth and shrinkage, a process known as dynamic instability. Understanding the mechanism of dynamic instability offers potential for controlling microtubule-dependent cellular processes such as nerve growth and mitosis. The growth to shrinkage transitions (catastrophes) and the reverse transitions (rescues) that characterize microtubule dynamic instability have been assumed to be random events with first-order kinetics. By direct observation of individual microtubules in vitro and probabilistic analysis of their distribution of growth times, we found that while the slower growing and biologically inactive (minus) ends obeyed first-order catastrophe kinetics, the faster growing and biologically active (plus) ends did not. The non-first-order kinetics at plus ends imply that growing microtubule plus ends have an effective frequency of catastrophe that depends on how long the microtubules have been growing. This frequency is low initially but then rises asymptotically to a limiting value. Our results also suggest that an additional parameter, beyond the four parameters typically used to describe dynamic instability, is needed to account for the observed behavior and that changing this parameter can significantly affect the distribution of microtubule lengths at steady state.

Asunto(s)

RESUMEN

The process of neurite outgrowth is critically dependent on proper microtubule assembly. However, characterizing the dynamics of microtubule assembly and their quantitative relationship to neurite outgrowth is a difficult task. The difficulty can be reduced by using time series analysis which has broad application in characterizing the dynamics of stochastic, or "noisy," behaviors. Here we apply time series analysis to quantitatively compare simulated microtubule assembly and neurite outgrowth in vitro. Microtubule length life histories were simulated assuming constant growth and shrinkage rates coupled with random selection of growth and shrinkage times, a formulation based on the dynamic instability model of microtubule assembly. Net length displacements of simulated microtubules were calculated at discrete, evenly spaced times, and the resulting time series were characterized by both spectral and autocorrelation analysis. Depending on the sampling rate and the dynamic parameters, simulated microtubules exhibited significant autocorrelation and periodicity. To make a comparison to neurite outgrowth, we characterized the dynamic behavior of simulated microtubule populations and found it was not significantly different from that of single microtubules. The net displacements of rat superior cervical ganglion neurite tips were measured and characterized using time series methods. Their behavior was consistent with the microtubule dynamics for appropriate simulation parameters and sampling rates. Our results show that time series analysis can provide a useful tool for quantitative characterization of microtubule dynamics and neurite outgrowth and for assessing the relationship between them.

Asunto(s)

RESUMEN

Immunoaffinity purification has become an important technique in biotechnology. In this review the basic principles of immunoaffinity separations are described with respect to the stages of operation and potential application. The most commonly used support materials, activation procedures, and coupling chemistries are compared to one another for suitability in various applications. Individual operational steps for fixed bed immunoadsorbents including loading, washing, elution and regeneration are described in terms of both theory and practice. Factors influencing adsorbent stability are identified, and alternative operation and configuration strategies are discussed in light of their application to immunoaffinity systems.