Raja Paul - Academia.edu (original) (raw)
Papers by Raja Paul
Cell, 2011
Error-free chromosome segregation requires stable attachment of sister kinetochores to the opposi... more Error-free chromosome segregation requires stable attachment of sister kinetochores to the opposite spindle poles (amphitelic attachment). Exactly how amphitelic attachments are achieved during spindle assembly remains elusive. We employed photoactivatable GFP and high-resolution live-cell confocal microscopy to visualize complete 3D movements of individual kinetochores throughout mitosis in nontransformed human cells. Combined with electron microscopy, molecular perturbations, and immunofluorescence analyses, this approach reveals unexpected details of chromosome behavior. Our data demonstrate that unstable lateral interactions between kinetochores and microtubules dominate during early prometaphase. These transient interactions lead to the reproducible arrangement of chromosomes in an equatorial ring on the surface of the nascent spindle. A computational model predicts that this toroidal distribution of chromosomes exposes kinetochores to a high density of microtubules which facilitates subsequent formation of amphitelic attachments. Thus, spindle formation involves a previously overlooked stage of chromosome prepositioning which promotes formation of amphitelic attachments.► In human cells, chromosomes form a ring around the spindle during prometaphase ► This arrangement requires chromokinesin-mediated ejection of chromosome arms ► Formation of the ring accelerates spindle assembly ► Stable amphitelic attachments form during late prometaphase and metaphase
Proceedings of The National Academy of Sciences, 2009
The mitotic spindle self-assembles in prometaphase by a combination of centrosomal pathway, in wh... more The mitotic spindle self-assembles in prometaphase by a combination of centrosomal pathway, in which dynamically unstable microtubules search in space until chromosomes are captured, and a chromosomal pathway, in which microtubules grow from chromosomes and focus to the spindle poles. Quantitative mechanistic understanding of how spindle assembly can be both fast and accurate is lacking. Specifically, it is unclear how, if at all, chromosome movements and combining the centrosomal and chromosomal pathways affect the assembly speed and accuracy. We used computer simulations and high-resolution microscopy to test plausible pathways of spindle assembly in realistic geometry. Our results suggest that an optimal combination of centrosomal and chromosomal pathways, spatially biased microtubule growth, and chromosome movements and rotations is needed to complete prometaphase in 10 -20 min while keeping erroneous merotelic attachments down to a few percent. The simulations also provide kinetic constraints for alternative error correction mechanisms, shed light on the dual role of chromosome arm volume, and compare well with experimental data for bipolar and multipolar HT-29 colorectal cancer cells.
Biophysical Journal, 2008
We investigate both theoretically and experimentally how stress is propagated through the actin c... more We investigate both theoretically and experimentally how stress is propagated through the actin cytoskeleton of adherent cells and consequentially distributed at sites of focal adhesions (FAs). The actin cytoskeleton is modeled as a twodimensional cable network with different lattice geometries. Both prestrain, resulting from actomyosin contractility, and central application of external force, lead to finite forces at the FAs that are largely independent of the lattice geometry, but strongly depend on the exact spatial distribution of the FAs. The simulation results compare favorably with experiments with adherent fibroblasts onto which lateral force is exerted using a microfabricated pillar. For elliptical cells, central application of external force along the long axis leads to two large stress regions located obliquely opposite to the pulling direction. For elliptical cells pulled along the short axis as well as for circular cells, there is only one region of large stress opposite to the direction of pull. If in the computer simulations FAs are allowed to rupture under force for elliptically elongated and circular cell shapes, then morphologies arise which are typical for migrating fibroblasts and keratocytes, respectively. The same effect can be obtained also by internally generated force, suggesting a mechanism by which cells can control their migration morphologies.
Adhesion-dependent soft tissue cells both create and sense tension in the extracellular matrix. T... more Adhesion-dependent soft tissue cells both create and sense tension in the extracellular matrix. Therefore cells can actively interact through the mechanics of the surrounding matrix. An intracellular positive feedback loop upregulates cellular contractility in stiff or tensed environments. Here we theoretically address the resulting pattern formation and force generation for the case of a filamentous matrix, which we model as a two-dimensional cable network. Cells are modeled as anisotropic contraction dipoles which move in favor of tensed directions in the matrix. Our Monte Carlo simulations suggest that at small densities, cells align in strings, while at high densities, they form interconnected meshworks. Cellular activation both by biochemical factors and by tension leads to a hyperbolic increase in tissue tension. We also discuss the effect of cell density on tissue tension and shape.
Cell, 2011
Error-free chromosome segregation requires stable attachment of sister kinetochores to the opposi... more Error-free chromosome segregation requires stable attachment of sister kinetochores to the opposite spindle poles (amphitelic attachment). Exactly how amphitelic attachments are achieved during spindle assembly remains elusive. We employed photoactivatable GFP and high-resolution live-cell confocal microscopy to visualize complete 3D movements of individual kinetochores throughout mitosis in nontransformed human cells. Combined with electron microscopy, molecular perturbations, and immunofluorescence analyses, this approach reveals unexpected details of chromosome behavior. Our data demonstrate that unstable lateral interactions between kinetochores and microtubules dominate during early prometaphase. These transient interactions lead to the reproducible arrangement of chromosomes in an equatorial ring on the surface of the nascent spindle. A computational model predicts that this toroidal distribution of chromosomes exposes kinetochores to a high density of microtubules which facilitates subsequent formation of amphitelic attachments. Thus, spindle formation involves a previously overlooked stage of chromosome prepositioning which promotes formation of amphitelic attachments.► In human cells, chromosomes form a ring around the spindle during prometaphase ► This arrangement requires chromokinesin-mediated ejection of chromosome arms ► Formation of the ring accelerates spindle assembly ► Stable amphitelic attachments form during late prometaphase and metaphase
Proceedings of The National Academy of Sciences, 2009
The mitotic spindle self-assembles in prometaphase by a combination of centrosomal pathway, in wh... more The mitotic spindle self-assembles in prometaphase by a combination of centrosomal pathway, in which dynamically unstable microtubules search in space until chromosomes are captured, and a chromosomal pathway, in which microtubules grow from chromosomes and focus to the spindle poles. Quantitative mechanistic understanding of how spindle assembly can be both fast and accurate is lacking. Specifically, it is unclear how, if at all, chromosome movements and combining the centrosomal and chromosomal pathways affect the assembly speed and accuracy. We used computer simulations and high-resolution microscopy to test plausible pathways of spindle assembly in realistic geometry. Our results suggest that an optimal combination of centrosomal and chromosomal pathways, spatially biased microtubule growth, and chromosome movements and rotations is needed to complete prometaphase in 10 -20 min while keeping erroneous merotelic attachments down to a few percent. The simulations also provide kinetic constraints for alternative error correction mechanisms, shed light on the dual role of chromosome arm volume, and compare well with experimental data for bipolar and multipolar HT-29 colorectal cancer cells.
Biophysical Journal, 2008
We investigate both theoretically and experimentally how stress is propagated through the actin c... more We investigate both theoretically and experimentally how stress is propagated through the actin cytoskeleton of adherent cells and consequentially distributed at sites of focal adhesions (FAs). The actin cytoskeleton is modeled as a twodimensional cable network with different lattice geometries. Both prestrain, resulting from actomyosin contractility, and central application of external force, lead to finite forces at the FAs that are largely independent of the lattice geometry, but strongly depend on the exact spatial distribution of the FAs. The simulation results compare favorably with experiments with adherent fibroblasts onto which lateral force is exerted using a microfabricated pillar. For elliptical cells, central application of external force along the long axis leads to two large stress regions located obliquely opposite to the pulling direction. For elliptical cells pulled along the short axis as well as for circular cells, there is only one region of large stress opposite to the direction of pull. If in the computer simulations FAs are allowed to rupture under force for elliptically elongated and circular cell shapes, then morphologies arise which are typical for migrating fibroblasts and keratocytes, respectively. The same effect can be obtained also by internally generated force, suggesting a mechanism by which cells can control their migration morphologies.
Adhesion-dependent soft tissue cells both create and sense tension in the extracellular matrix. T... more Adhesion-dependent soft tissue cells both create and sense tension in the extracellular matrix. Therefore cells can actively interact through the mechanics of the surrounding matrix. An intracellular positive feedback loop upregulates cellular contractility in stiff or tensed environments. Here we theoretically address the resulting pattern formation and force generation for the case of a filamentous matrix, which we model as a two-dimensional cable network. Cells are modeled as anisotropic contraction dipoles which move in favor of tensed directions in the matrix. Our Monte Carlo simulations suggest that at small densities, cells align in strings, while at high densities, they form interconnected meshworks. Cellular activation both by biochemical factors and by tension leads to a hyperbolic increase in tissue tension. We also discuss the effect of cell density on tissue tension and shape.