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David J Odde

Publications and source records attributed to David J Odde.

At least 19 recordsLinked to original sources
PubMed2007

Hypothesis testing via integrated computer modeling and digital fluorescence microscopy.

Computational modeling has the potential to add an entirely new approach to hypothesis testing in yeast cell biology. Here, we present a method for seamless integration of computational modeling with quantitative digital fluorescence microscopy. This integration is accomplished by developing computational models based on hypotheses for underlying cellular processes that may give rise to experimentally observed fluorescent protein localization patterns. Simulated fluorescence images are generated from the computational models of underlying cellular processes via a "model-convolution" process. These simulated images can then be directly compared to experimental fluorescence images in order to test the model. This method provides a framework for rigorous hypothesis testing in yeast cell biology via integrated mathematical modeling and digital fluorescence microscopy.

Computational Biology↗
PubMed2006

Asymmetric division: motor persistence pays off.

A new study shows that an antagonistic force model can explain a number of complex mitotic spindle movements in the first mitosis of the Caenorhabditis elegans embryo by simply assuming that cortical force generators become increasingly persistent in their interaction with microtubules during mitosis.

Animals↗
PubMed2006

Potential for control of signaling pathways via cell size and shape.

BACKGROUND: In order for signals generated at the plasma membrane to reach intracellular targets, activated messengers, such as G proteins and phosphoproteins, must diffuse through the cytoplasm. If the deactivators of these messengers, GTPase activating proteins (GAPs) and phosphatases, respectively, are sufficiently active in the cytoplasm, then the signal could in principle decay before reaching the target and a stable spatial gradient in phosphostate would be generated. Recent experiments document the existence of such gradients in living cells and suggest a role for them in mitotic spindle morphogenesis and cell migration. However, how such systems behave theoretically when embedded in a cell of varying size or shape has not been considered. RESULTS: Here we use a simple mathematical model to explore the theoretical consequences of a plasma membrane bound activator (i.e., guanine nucleotide exchange factor, GEF, or kinase) and a cytoplasmic deactivator (i.e., GAP or phosphatase), and we find that as a model cell grows, the substrate becomes progressively dephosphorylated as a result of decreased proximity to the activator. Conversely, as a cell spreads and flattens, the substrate becomes globally phosphorylated because of increased proximity of the substrate to the activator. Similarly, in the leading edge of polarized cells and in protrusions such as lamellipodia or filopodia, the substrate is highly phosphorylated. As a specific test of the model, we found that the experimentally observed preferential activation of the G protein Cdc42 in the periphery of fibroblasts that was recently reported is consistent with model predictions. CONCLUSIONS: We conclude that cell-signaling pathways can theoretically be turned on and off, both locally and globally, in response to alterations in cell size and shape.

Cell Shape↗
PubMed2006

Mps1 phosphorylation of Dam1 couples kinetochores to microtubule plus ends at metaphase.

BACKGROUND: Duplicated chromosomes are equally segregated to daughter cells by a bipolar mitotic spindle during cell division. By metaphase, sister chromatids are coupled to microtubule (MT) plus ends from opposite poles of the bipolar spindle via kinetochores. Here we describe a phosphorylation event that promotes the coupling of kinetochores to microtubule plus ends. RESULTS: Dam1 is a kinetochore component that directly binds to microtubules. We identified DAM1-765, a dominant allele of DAM1, in a genetic screen for mutations that increase stress on the spindle pole body (SPB) in Saccharomyces cerevisiae. DAM1-765 contains the single mutation S221F. We show that S221 is one of six Dam1 serines (S13, S49, S217, S218, S221, and S232) phosphorylated by Mps1 in vitro. In cells with single mutations S221F, S218A, or S221A, kinetochores in the metaphase spindle form tight clusters that are closer to the SPBs than in a wild-type cell. Five lines of experimental evidence, including localization of spindle components by fluorescence microscopy, measurement of microtubule dynamics by fluorescence redistribution after photobleaching, and reconstructions of three-dimensional structure by electron tomography, combined with computational modeling of microtubule behavior strongly indicate that, unlike wild-type kinetochores, Dam1-765 kinetochores do not colocalize with an equal number of plus ends. Despite the uncoupling of the kinetochores from the plus ends of MTs, the DAM1-765 cells are viable, complete the cell cycle with the same kinetics as wild-type cells, and biorient their chromosomes as efficiently as wild-type cells. CONCLUSIONS: We conclude that phosphorylation of Dam1 residues S218 and S221 by Mps1 is required for efficient coupling of kinetochores to MT plus ends. We find that efficient plus-end coupling is not required for (1) maintenance of chromosome biorientation, (2) maintenance of tension between sister kinetochores, or (3) chromosome segregation.

Cell Cycle Proteins↗
PubMed2006

Measuring nanometer scale gradients in spindle microtubule dynamics using model convolution microscopy.

A computational model for the budding yeast mitotic spindle predicts a spatial gradient in tubulin turnover that is produced by kinetochore-attached microtubule (kMT) plus-end polymerization and depolymerization dynamics. However, kMTs in yeast are often much shorter than the resolution limit of the light microscope, making visualization of this gradient difficult. To overcome this limitation, we combined digital imaging of fluorescence redistribution after photobleaching (FRAP) with model convolution methods to compare computer simulations at nanometer scale resolution to microscopic data. We measured a gradient in microtubule dynamics in yeast spindles at approximately 65-nm spatial intervals. Tubulin turnover is greatest near kinetochores and lowest near the spindle poles. A beta-tubulin mutant with decreased plus-end dynamics preserves the spatial gradient in tubulin turnover at a slower time scale, increases average kinetochore microtubule length approximately 14%, and decreases tension at kinetochores. The beta-tubulin mutant cells have an increased frequency of chromosome loss, suggesting that the accuracy of chromosome segregation is linked to robust kMT plus-end dynamics.

Chromosome Segregation↗
PubMed2006

Two-step cell patterning on planar and complex curved surfaces by precision spraying of polymers.

Controlling adhesion of living animal cells plays a key role in biosensor fabrication, drug-testing technologies, basic biological research, and tissue engineering applications. Current techniques for cell patterning have two primary limitations: (1) they require photolithography, and (2) they are limited to patterning of planar surfaces. Here we demonstrate a simple, precision spraying method for both positive and negative patterning of planar and curved surfaces to achieve cell patterns rapidly and reproducibly. In this method, which we call precision spraying (PS), a polymer solution is aerosolized, focused with sheath airflow through an orifice, and deposited on the substrate using a deposition head to create approximately 25 microm sized features. In positive patterning, adhesive molecules, such as laminin or polyethylenimine (PEI) were patterned on polydimethylsiloxane (PDMS) substrates in a single spraying operation. A variety of animal cell types were found to adhere to the adhesive regions, and avoid the non-adhesive (bare PDMS) regions. In negative patterning, hydrophobic materials, such as polytetrafluoroethylene (PTFE) and PDMS, were patterned on glass substrates. Cells then formed patterns on the exposed glass regions and avoided the hydrophobic regions. Cellular patterns were maintained for up to 2 weeks in the presence of serum, which normally fouls non-adhesive regions. Additionally, we found that precision spraying enabled micropatterning of complex-curved surfaces. Our results show that precision spraying followed by cell plating enables rapid and flexible cellular micropatterning in two simple steps.

Animals↗
PubMed2006

Modeling of chromosome motility during mitosis.

Chromosome motility is a highly regulated and complex process that ultimately achieves proper segregation of the replicated genome. Recent modeling studies provide a computational framework for investigating how microtubule assembly dynamics, motor protein activity and mitotic spindle mechanical properties are integrated to drive chromosome motility. Among other things, these studies show that metaphase chromosome oscillations can be explained by a range of assumptions, and that non-oscillatory states can be achieved with modest changes to the model parameters. In addition, recent microscopy studies provide new insight into the nature of the coupling between force on the kinetochore and kinetochore-microtubule assembly/disassembly. Together, these studies facilitate advancement toward a unified model that quantitatively predicts chromosome motility.

Animals↗
PubMed2006

Endothelium-mediated hepatocyte recruitment in the establishment of liver-like tissue in vitro.

A major goal of liver tissue engineering is to understand how the constituent cell types interact to achieve liver-specific structure and function. Here we show that hepatocytes migrate toward and adhere to endothelial vascular structures formed on Matrigel in vitro, and that hepatocyte recruitment is dependent on endothelium-derived hepatocyte growth factor. The hepatocyte-decorated endothelial vascular structures resemble In vivo sinusoids containing plate-like structures, bile canaliculi, and a lumen. The sinusoid-like structures retained cytochrome P450 expression and activity, in addition to stable albumin expression and secretion rate for over 2 months in vitro. The stability of the sinusoid-like structures was dependent on the presence of vimentin-positive fibroblasts in culture. The sinusoid-like structures formed by hepatocytes and pure populations of endothelial cells collapsed after 10 days in culture. In contrast, culture of hepatocytes with fibroblast-contaminated human dermal microvascular endothelial cells or a combination of human umbilical vein endothelial cells and normal human dermal fibroblasts resulted in stable sinusoid-like structures surrounded by a fibroblastic capsule that maintained liver specific functions for several months in vitro. These results demonstrate that specification of endothelial cell position ultimately determines hepatocyte position in vitro, suggesting that similar interactions might occur In vivo. The novelty of the culture's sinusoid-like organization and long-term function suggest a new model for the study of liver toxicity, ischemia/reperfusion injury, and fibrosis.

Adult↗
PubMed2005

Mitotic spindle: disturbing a subtle balance.

Mitotic spindles maintain a roughly constant length in metaphase, so the forces between the spindle poles are balanced. A new study involving screening molecules believed to mediate this force balance has found that spindle length is relatively insensitive to perturbations of molecular motor force-generating activities, but more sensitive to perturbation of microtubule assembly regulators and chromosome cohesion.

Animals↗
PubMed2005

Laser-guided direct writing for three-dimensional tissue engineering.

One of the principal limitations to the size of an engineered tissue is oxygen and nutrient transport. Lacking a vascular bed, cells embedded in an engineered tissue will consume all available oxygen within hours while out branching blood vessels will take days to vascularize the implanted tissue. One possible solution is to directly write vascular structures within the engineered tissue prior to implantation, reconstructing the tissue according to its native architecture. The cell patterning technique, laser-guided direct writing (LGDW), can pattern multiple cells types with micrometer resolution on arbitrary surfaces, including biological gels. Here we show that LGDW can pattern human umbilical vein endothelial cells (HUVEC) in two- and three-dimensions with micrometer accuracy. By patterning HUVEC on Matrigel, we can direct their self-assembly into vascular structures along the desired pattern. Finally, co-culturing the vascular structures with hepatocytes resulted in an aggregated tubular structure similar in organization to a hepatic sinusoid. This capability can facilitate studies of tissue architecture at the single cell level, and of heterotypic interactions underlying processes such as liver and pancreas morphogenesis, differentiation, and angiogenesis.

Animals↗
PubMed2005

Mechanochemical model of microtubule structure and self-assembly kinetics.

Microtubule self-assembly is largely governed by the chemical kinetics and thermodynamics of tubulin-tubulin interactions. An important aspect of microtubule assembly is that hydrolysis of the beta-tubulin-associated GTP promotes protofilament curling. Protofilament curling presumably drives the transition from tip structures associated with growth (sheetlike projections and blunt ends) to those associated with shortening (rams' horns and frayed ends), and transitions between these structures have been proposed to be important for growth-shortening transitions. However, previous models for microtubule dynamic instability have not considered such structures or mechanics explicitly. Here we present a three-dimensional model that explicitly incorporates mechanical stress and strain within the microtubule lattice. First, we found that the model recapitulates three-dimensional tip structures and rates of assembly and disassembly for microtubules grown under standard conditions, and we propose that taxol may stabilize microtubule growth by reducing flexural rigidity. Second, in contrast to recent suggestions, it was determined that sheetlike tips are more likely to undergo catastrophe than blunt tips. Third, partial uncapping of the tubulin-GTP cap provides a possible mechanism for microtubule pause events. Finally, simulations of the binding and structural effects of XMAP215 produced the experimentally observed growth and shortening rates, and tip structure.

Biophysics↗
PubMed2005

Tension-dependent regulation of microtubule dynamics at kinetochores can explain metaphase congression in yeast.

During metaphase in budding yeast mitosis, sister kinetochores are tethered to opposite poles and separated, stretching their intervening chromatin, by singly attached kinetochore microtubules (kMTs). Kinetochore movements are coupled to single microtubule plus-end polymerization/depolymerization at kinetochore attachment sites. Here, we use computer modeling to test possible mechanisms controlling chromosome alignment during yeast metaphase by simulating experiments that determine the 1) mean positions of kinetochore Cse4-GFP, 2) extent of oscillation of kinetochores during metaphase as measured by fluorescence recovery after photobleaching (FRAP) of kinetochore Cse4-GFP, 3) dynamics of kMTs as measured by FRAP of GFP-tubulin, and 4) mean positions of unreplicated chromosome kinetochores that lack pulling forces from a sister kinetochore. We rule out a number of possible models and find the best fit between theory and experiment when it is assumed that kinetochores sense both a spatial gradient that suppresses kMT catastrophe near the poles and attachment site tension that promotes kMT rescue at higher amounts of chromatin stretch.

Cell Cycle Proteins↗
PubMed2005

Chromosome capture: take me to your kinetochore.

To form a proper mitotic spindle, all kinetochores must capture at least one microtubule plus end. A new computational modeling study shows that a search-and-capture mechanism based on spatially unbiased microtubule dynamic instability is too slow to account for the experimentally observed rate of chromosome capture.

Chromosomes↗
PubMed2005

Robust micromechanical neurite elicitation in synapse-competent neurons via magnetic bead force application.

The ability to engineer living networks of interconnected neurons with specified connectivity would facilitate the study of synaptogenesis and information processing in the nervous system. Previously, we found that a neurite can be elicited from embryonic chick forebrain neurons by direct mechanical means using magnetic bead force application (MBFA); however, our previous studies and others focused on young, synapse-incompetent neurons. To address this issue, we tested cultures of embryonic chick forebrain neurons of varying age and found that neurites could be micromechanically elicited via MBFA at all ages tested, which ranged between 7 and 22 embryonic equivalent (EE) days (days in ovo plus days in vitro). The probability of neurite initiation was at least 40% for all ages, with a maximum of approximately 80% after 2-4 days in vitro, and a decrease to approximately 60% by day 10 in vitro. The force required to elicit a neurite was approximately 1500 pN with a minimum of approximately 700 pN at embryonic equivalent day 14. The probability of success was similar for two rates of force application (10 and 500 pN/s). Neurite initiation via micromechanical force is robust with respect to cell age, and micromechanical force can induce neurites in synapse-competent neurons.

Animals↗
PubMed2004

Dimensionless parameters for the design of optical traps and laser guidance systems.

Optical traps are routinely used for the manipulation of neutral particles. However, optical trap design is limited by the lack of an accurate theory. The generalized Lorenz-Mie theory (GLMT) solves the scattering problem for arbitrary particle size and predicts radial forces accurately. Here we show that the GLMT predicts the observed radial and axial forces in a variety of optical manipulators. We also present a dimensionless parameter beta for the prediction of axial forces. Coupled with our correlation for radial escape forces, we now have a set of two simple correlations for the practical design of radiation-force-based systems.

Algorithms↗
PubMed2003

Tensile force-dependent neurite elicitation via anti-beta1 integrin antibody-coated magnetic beads.

Previous work using glass microneedles to apply calibrated, localized force to neurons showed that tensile force is a sufficient signal for neurite initiation and elongation. However, previous studies did not examine the kinetics or probability of neurite initiation as a function of force or the rate of force application. Here we report the use of a new technique-magnetic bead force application-to systematically investigate the role of force in these phenomena with better ease of use and control over force than glass microneedles. Force-induced neurite initiation from embryonic chick forebrain neurons appeared to be a first-order random process whose rate increased with increasing force, and required the presence of peripheral microtubules. In addition, the probability of initiation was more than twofold lower for neurons exposed to rapid initial force ramps (450 pN/s) than for neurons exposed to slower ramps (1.5 and 11 pN/s). We observed a low force threshold for elongation (15-100 pN), which was not previously detected in chick forebrain neurites elongated by glass microneedles. Finally, neurites subjected to constant force elongated at variable instantaneous rates, and switched abruptly between elongation and retraction, similar to spontaneous, growth-cone-mediated outgrowth and microtubule dynamic instability.

Animals↗
PubMed2003

Mechanisms of microtubule-based kinetochore positioning in the yeast metaphase spindle.

It has been hypothesized that spatial gradients in kMT dynamic instability facilitate mitotic spindle formation and chromosome movement. To test this hypothesis requires the analysis of kMT dynamics, which have not been resolved at the single kMT level in living cells. The budding yeast spindle offers an attractive system in which to study kMT dynamics because, in contrast to animal cells, there is only one kMT per kinetochore. To visualize metaphase kMT plus-end dynamics in yeast, a strain containing a green fluorescent protein fusion to the kinetochore protein, Cse4, was imaged by fluorescence microscopy. Although individual kinetochores were not resolvable, we found that models of kMT dynamics could be evaluated by simulating the stochastic kMT dynamics and then simulating the fluorescence imaging of kMT plus-end-associated kinetochores. Statistical comparison of model-predicted images to experimentally observed images demonstrated that a pure dynamic instability model for kMT dynamics in the yeast metaphase spindle was unacceptable. However, when a temporally stable spatial gradient in the catastrophe or rescue frequency was added to the model, there was reasonable agreement between the model and the experiment. These results provide the first evidence of temporally stable spatial gradients of kMT catastrophe and/or rescue frequency in living cells.

Cell Movement↗