Cellular Dynamics Program
Permanent URI for this collection
The Cellular Dynamics Program (CDP) at the MBL aims to accelerate the knowledge of basic biology and disease through the development and application of biophysical methods customized to shed light on life’s most essential processes.
CDP is composed of several independent laboratories, notably those with a specialization in imaging and cell physiology and biochemistry. Additionally the program houses the NIH:NCRR national resource, the BioCurrents Research Center. The CDP imaging component focuses on the architectural dynamics of living cells, which encompass the timely and coordinated assembly and disassembly of macromolecular structures essential for the proper functioning and differentiation of cells, the spatial and temporal organization of these structures, and their physiological and genetic control. The molecular physiology component, in pursuing studies of cell metabolism and transport biophysics, has pioneered the use of electrochemical sensors to define the chemical signatures surrounding living cells and tissues, opening insights to cell function from a distance.
Browse
Browsing Cellular Dynamics Program by Issue Date
Results Per Page
Sort Options
-
ArticleA fiber-optic phase-randomizer for microscope illumination by laser(Rockefeller University Press, 1979-11) Ellis, Gordon W.
-
Moving ImageTime lapse movie of meiosis II in a living spermatocyte from the crane fly, Nephrotoma suturalis, viewed with polarized light microscopy( 2002-11) LaFountain, James R. ; Oldenbourg, RudolfThe events of meiosis II in two living spermatocytes obtained from the testis of a crane-fly larva are recorded in this time-lapse sequence beginning at prophase II through telophase II to the near completion of cytokinesis following meiosis II.
-
Moving ImageTime lapse movie of meiosis I in a living spermatocyte from the crane fly, Nephrotoma suturalis, viewed with polarized light microscopy( 2002-11) LaFountain, James R. ; Oldenbourg, RudolfThe events of meiosis I in a living spermatocyte obtained from the testis of a crane-fly larva are recorded in this time-lapse sequence beginning at diakinesis through telophase to the near completion of cytokinesis following meiosis I.
-
Book chapterPolarization microscopy with the LC-PolScope( 2003-11) Oldenbourg, RudolfIn the current chapter we describe the use of a new type of polarized light microscope which we started to develop at the Marine Biological Laboratory about ten years ago. The new “PolScope” is based on the traditional polarized light microscope and enhances it with the use of liquid- crystal devices and special image processing algorithms. The LC-PolScope measures polarization optical parameters in many specimen points simultaneously, in fast time intervals, and at the highest resolution of the light microscope. It rapidly generates a birefringence map whose pixel brightness is directly proportional to the local optical anisotropy, unaffected by the specimen orientation in the plane of view, as well as a map depicting the slow axis orientation of the birefringent regions. The basic LC-PolScope technology can be adapted to most research grade microscopes and is available commercially from Cambridge Research and Instrumentation (CRI, http://www.cri-inc.com) in Woburn, Massachusetts, under the trade name LC-PolScope.
-
ArticleMicrotubule plus-end dynamics in Xenopus egg extract spindles(American Society for Cell Biology, 2004-02-06) Tirnauer, Jennifer S. ; Salmon, Edward D. ; Mitchison, Timothy J.Microtubule dynamics underlie spindle assembly, yet we do not know how the spindle environment affects these dynamics. We developed methods for measuring two key parameters of microtubule plus-end dynamic instability in Xenopus egg extract spindles. To measure plus-end polymerization rates and localize growing plus ends, we used fluorescence confocal imaging of EB1. This revealed plus-end polymerization throughout the spindle at ~11 µm/min, similar to astral microtubules, suggesting polymerization velocity is not regionally regulated by the spindle. The ratio of EB1 to microtubule fluorescence revealed an enrichment of polymerizing ends near the spindle middle, indicating enhanced nucleation or rescue there. We measured depolymerization rates by creating a front of synchronized depolymerization in spindles severed with microneedles. This front could be tracked by polarization and fluorescence microscopy as it advanced from each cut edge toward the associated pole. Both imaging modalities revealed rapid depolymerization (~30 µm/min) superimposed on a subset of microtubules stable to depolymerization. Larger spindle fragments contained a higher percentage of stable microtubules, which we believe were oriented with their minus ends facing the cut. Depolymerization was blocked by the potent microtubule stabilizing agent hexylene glycol, but was unaffected by {alpha}-MCAK antibody and AMPPNP, which block catastrophe and kinesin motility, respectively. These measurements move us closer to understanding the complete life history of a spindle microtubule.
-
ArticleBipolarization and poleward flux correlate during xenopus extract spindle assembly(American Society for Cell Biology, 2004-09-22) Mitchison, Timothy J. ; Maddox, P. ; Groen, Aaron C. ; Cameron, Lisa ; Perlman, Z. ; Ohi, Ryoma ; Desai, Ankur R. ; Salmon, Edward D. ; Kapoor, Tarun M.We investigated the mechanism by which meiotic spindles become bipolar and the correlation between bipolarity and poleward flux, using Xenopus egg extracts. By speckle microscopy and computational alignment, we find that monopolar sperm asters do not show evidence for flux, partially contradicting previous work. We account for the discrepancy by describing spontaneous bipolarization of sperm asters that was missed previously. During spontaneous bipolarization, onset of flux correlated with onset of bipolarity, implying that antiparallel microtubule organization may be required for flux. Using a probe for TPX2 in addition to tubulin, we describe two pathways that lead to spontaneous bipolarization, new pole assembly near chromatin, and pole splitting. By inhibiting the Ran pathway with excess importin-alpha, we establish a role for chromatin-derived, antiparallel overlap bundles in generating the sliding force for flux, and we examine these bundles by electron microscopy. Our results highlight the importance of two processes, chromatin-initiated microtubule nucleation, and sliding forces generated between antiparallel microtubules, in self-organization of spindle bipolarity and poleward flux.
-
ArticleMaloriented bivalents have metaphase positions at the spindle equator with more kinetochore microtubules to one pole than to the other(American Society for Cell Biology, 2004-09-22) LaFountain, James R. ; Oldenbourg, RudolfTo test the "traction fiber" model for metaphase positioning of bivalents during meiosis, kinetochore fibers of maloriented bivalents, induced during recovery from cold arrest, were analyzed with a liquid crystal polarizing microscope. The measured birefringence retardation of kinetochore fibers is proportional to the number of microtubules in a fiber. Five of the 11 maloriented bivalents analyzed exhibited bipolar malorientations that had at least four times more kinetochore microtubules to one pole than to the other pole, and two had microtubules directed to only one pole. Yet all maloriented bivalents had positions at or near the spindle equator. The traction fiber model predicts such maloriented bivalents should be positioned closer to the pole with more kinetochore microtubules. A metaphase position at the spindle equator, according to the model, requires equal numbers of kinetochore microtubules to both poles. Data from polarizing microscope images were not in accord with those predictions, leading to the conclusion that other factors, in addition to traction forces, must be involved in metaphase positioning in crane-fly spermatocytes. Although the identity of additional factors has not been established, one possibility is that polar ejection forces operate to exert away-from-the-pole forces that could counteract pole-directed traction forces. Another is that kinetochores are "smart," meaning they embody a position-sensitive mechanism that controls their activity.
-
ArticleRoles of polymerization dynamics, opposed motors, and a tensile element in governing the length of Xenopus extract meiotic spindles(American Society for Cell Biology, 2005-03-23) Mitchison, Timothy J. ; Maddox, P. ; Gaetz, J. ; Groen, Aaron C. ; Shirasu, M. ; Desai, Ankur R. ; Salmon, Edward D. ; Kapoor, Tarun M.Metaphase spindles assemble to a steady state in length by mechanisms that involve microtubule dynamics and motor proteins, but they are incompletely understood. We found that Xenopus extract spindles recapitulate the length of egg meiosis II spindles, by using mechanisms intrinsic to the spindle. To probe these mechanisms, we perturbed microtubule polymerization dynamics and opposed motor proteins and measured effects on spindle morphology and dynamics. Microtubules were stabilized by hexylene glycol and inhibition of the catastrophe factor mitotic centromere-associated kinesin (MCAK) (a kinesin 13, previously called XKCM) and destabilized by depolymerizing drugs. The opposed motors Eg5 and dynein were inhibited separately and together. Our results are consistent with important roles for polymerization dynamics in regulating spindle length, and for opposed motors in regulating the relative stability of bipolar versus monopolar organization. The response to microtubule destabilization suggests that an unidentified tensile element acts in parallel with these conventional factors, generating spindle shortening force.
-
PreprintChromosomes can congress to the metaphase plate before biorientation( 2005-12-09) Kapoor, Tarun M. ; Lampson, Michael A. ; Hergert, Polla ; Cameron, Lisa ; Cimini, Daniela ; Salmon, Edward D. ; McEwen, Bruce F. ; Khodjakov, AlexeyThe stable propagation of genetic material during cell division depends on the congression of chromosomes to the spindle equator before the cell initiates anaphase. It is generally assumed that congression requires that chromosomes are connected to the opposite poles of the bipolar spindle (i.e., “bi-oriented”). We found that chromosomes can congress before becoming bioriented. By combining the use of reversible chemical inhibitors, live-cell light microscopy and correlative electron microscopy, we found that mono-oriented chromosomes could glide towards the spindle equator alongside kinetochore fibers attached to other already bi-oriented chromosomes. This congression mechanism depended on the kinetochore-associated plus enddirected microtubule motor CENP-E (kinesin-7).
-
PreprintOrientation-independent differential interference contrast microscopy( 2006) Shribak, Michael ; Inoue, ShinyaThe image in a regular DIC microscope reflects the orientation of the prism shear direction and the optical path gradients in a phase specimen. If the shear direction lies parallel to the specimen boundary no contrast is generated. Also a bias retardance is generally introduced, which creates a gray background and reduces image contrast. Here we describe the theoretical foundation for a new DIC technique, which records phase gradients independently of their orientation and with the digitally generated gradient magnitude image as well as the optical path distribution image free from the gray background. Separate images can show the magnitude distribution of the optical path gradients and of the azimuths, or the two images can be combined into one picture e.g., with the brightness representing magnitudes and color showing azimuths respectively. For experimental verification of the proposed technique we investigated various specimens such as glass rods embedded in Permount, Siemens star nano-fabricated in 90-nm thick silicon oxide layer, Bovine pulmonary artery endothelial cell, etc, using regular DIC optics on a microscope equipped with a precision rotating stage. Several images were recorded with the specimen oriented in different directions, but with the prism bias unchanged, followed by digital alignment and processing of the images. The results demonstrate that the proposed DIC technique can successfully image and measure phase gradients of transparent specimens, independent of the directions of the gradient. The orientation-independent DIC data obtained can also be used to compute the quantitative distribution of specimen phase or to generate enhanced, regular DIC images with any desired shear direction. We are currently developing a new device using special DIC prisms, which allows the bias and shear directions to be switched rapidly without the need to mechanically rotate the specimen or the prism (US Patent Application 2005-0152030). With the new system an orientation independent DIC image should be obtained in a fraction of a second. A detailed description of the new system will be given in a future publication.
-
ArticleEntropy-driven formation of a chiral liquid-crystalline phase of helical filaments(American Physical Society, 2006-01-11) Barry, Edward ; Hensel, Zach ; Dogic, Zvonimir ; Shribak, Michael ; Oldenbourg, RudolfWe study the liquid-crystalline phase behavior of a concentrated suspension of helical flagella isolated from Salmonella typhimurium. Flagella are prepared with different polymorphic states, some of which have a pronounced helical character while others assume a rodlike shape. We show that the static phase behavior and dynamics of chiral helices are very different when compared to simpler achiral hard rods. With increasing concentration, helical flagella undergo an entropy-driven first order phase transition to a liquid-crystalline state having a novel chiral symmetry.
-
PreprintSelf-organization of actin filament orientation in the dendritic-nucleation/array-treadmilling model( 2006-11-16) Schaus, Thomas E. ; Taylor, Edwin W. ; Borisy, Gary G.The dendritic-nucleation/array-treadmilling model provides a conceptual framework for the generation of the actin network driving motile cells. We have incorporated it into a 2-D, stochastic computer model to study lamellipodia via the self-organization of filament orientation patterns. Essential dendritic-nucleation sub-models were incorporated, including discretized actin monomer diffusion, Monte-Carlo filament kinetics, and flexible filament and plasma membrane mechanics. Model parameters were estimated from the literature and simulation, providing values for the extent of the leading edge branching/capping-protective zone (5.4 nm) and the auto-catalytic branch rate (0.43 /s). For a given set of parameters the system evolved to a steady state filament count and velocity, at which total branching and capping rates were equal only for specific orientations; net capping eliminated others. The standard parameter set evoked a sharp preference for the ±35 deg. filaments seen in lamellipodial electron micrographs, requiring ~ 12 generations of successive branching to adapt to a 15 deg. change in protrusion direction. This pattern was robust with respect to membrane surface and bending energies and to actin concentrations, but required protection from capping at the leading edge and branching angles greater than 60 deg. A +70/0/-70 deg. pattern was formed with flexible filaments ~ 100 nm or longer and with velocities less than ~ 20% of free polymerization rates.
-
ArticleChromosome malorientations after meiosis II arrest cause nondisjunction(American Society for Cell Biology, 2007-02-21) Janicke, Marie A. ; Lasko, Loren ; Oldenbourg, Rudolf ; LaFountain, James R.This study investigated the basis of meiosis II nondisjunction. Cold arrest induced a fraction of meiosis II crane fly spermatocytes to form (n + 1) and (n – 1) daughters during recovery. Live-cell liquid crystal polarized light microscope imaging showed nondisjunction was caused by chromosome malorientation. Whereas amphitely (sister kinetochore fibers to opposite poles) is normal, cold recovery induced anaphase syntely (sister fibers to the same pole) and merotely (fibers to both poles from 1 kinetochore). Maloriented chromosomes had stable metaphase positions near the equator or between the equator and a pole. Syntelics were at the spindle periphery at metaphase; their sisters disconnected at anaphase and moved all the way to a centrosome, as their strongly birefringent kinetochore fibers shortened. The kinetochore fibers of merotelics shortened little if any during anaphase, making anaphase lag common. If one fiber of a merotelic was more birefringent than the other, the less birefringent fiber lengthened with anaphase spindle elongation, often permitting inclusion of merotelics in a daughter nucleus. Meroamphitely (near amphitely but with some merotely) caused sisters to move in opposite directions. In contrast, syntely and merosyntely (near syntely but with some merotely) resulted in nondisjunction. Anaphase malorientations were more frequent after longer arrests, with particularly long arrests required to induce syntely and merosyntely.
-
ArticleEna/VASP proteins have an anti-capping independent function in filopodia formation(American Society for Cell Biology, 2007-05-02) Applewhite, Derek A. ; Barzik, Melanie ; Kojima, Shin-ichiro ; Svitkina, Tatyana M. ; Gertler, Frank B. ; Borisy, Gary G.Filopodia have been implicated in a number of diverse cellular processes including growth-cone path finding, wound healing, and metastasis. The Ena/VASP family of proteins has emerged as key to filopodia formation but the exact mechanism for how they function has yet to be fully elucidated. Using cell spreading as a model system in combination with small interfering RNA depletion of Capping Protein, we determined that Ena/VASP proteins have a role beyond anticapping activity in filopodia formation. Analysis of mutant Ena/VASP proteins demonstrated that the entire EVH2 domain was the minimal domain required for filopodia formation. Fluorescent recovery after photobleaching data indicate that Ena/VASP proteins rapidly exchange at the leading edge of lamellipodia, whereas virtually no exchange occurred at filopodial tips. Mutation of the G-actin–binding motif (GAB) partially compromised stabilization of Ena/VASP at filopodia tips. These observations led us to propose a model where the EVH2 domain of Ena/VASP induces and maintains clustering of the barbed ends of actin filaments, which putatively corresponds to a transition from lamellipodial to filopodial localization. Furthermore, the EVH1 domain, together with the GAB motif in the EVH2 domain, helps to maintain Ena/VASP at the growing barbed ends.
-
ArticleIntrinsic dynamic behavior of fascin in filopodia(American Society for Cell Biology, 2007-08-01) Aratyn, Yvonne S. ; Schaus, Thomas E. ; Taylor, Edwin W. ; Borisy, Gary G.Recent studies showed that the actin cross-linking protein, fascin, undergoes rapid cycling between filopodial filaments. Here, we used an experimental and computational approach to dissect features of fascin exchange and incorporation in filopodia. Using expression of phosphomimetic fascin mutants, we determined that fascin in the phosphorylated state is primarily freely diffusing, whereas actin bundling in filopodia is accomplished by fascin dephosphorylated at serine 39. Fluorescence recovery after photobleaching analysis revealed that fascin rapidly dissociates from filopodial filaments with a kinetic off-rate of 0.12 s–1 and that it undergoes diffusion at moderate rates with a coefficient of 6 µm2s–1. This kinetic off-rate was recapitulated in vitro, indicating that dynamic behavior is intrinsic to the fascin cross-linker. A computational reaction–diffusion model showed that reversible cross-linking is required for the delivery of fascin to growing filopodial tips at sufficient rates. Analysis of fascin bundling indicated that filopodia are semiordered bundles with one bound fascin per 25–60 actin monomers.
-
ArticleQuantitative orientation-independent differential interference contrast (DIC) microscopy coupled with orientation-independent Polarization microscopy(Cambridge University Press, 2007-08-05) Shribak, Michael ; LaFountain, James R. ; Biggs, David ; Inoue, ShinyaDifferential interference contrast (DIC) microscopy is widely used to observe structure and motion in unstained, transparent living cells and isolated organelles, producing a monochromatic shadowcast image of optical phase gradient. Polarized light microscopy (Pol) reveals structural anisotropy due to form birefringence, intrinsic birefringence, stress birefringence, etc. DIC and Pol complement each other as, for example, in a live dividing cell, the DIC image will clearly show the chromosomes while the Pol image will depict the distribution of the birefringent microtubules in the spindle. Both methods, however, have the same shortcomings: they require the proper orientation of a specimen in relation to the optical system in order to achieve best results.
-
PreprintMicrotubule dynamics in cell division : exploring living cells with polarized light microscopy( 2008-04-11) Inoue, ShinyaThis Perspective is an account of my early experience while I studied the dynamic organization and behavior of the mitotic spindle and its submicroscopic filaments using polarized light microscopy. The birefringence of spindle filaments in normally dividing plant and animal cells, and those treated by various agents, revealed: A) the reality of spindle fibers and fibrils in healthy living cells; B) the labile, dynamic nature of the molecular filaments making up the spindle fibers; C) the mode of fibrogenesis and action of orienting centers; and D) force-generating properties based on the disassembly and assembly of the fibrils. These studies, which were carried out directly on living cells using improved polarizing microscopes, in fact, predicted the reversible assembly properties of isolated microtubules.
-
ArticleCalyculin-A induces cleavage in a random plane in unfertilized sea urchin eggs(Marine Biological Laboratory, 2009-02) Goda, Makoto ; Inoue, Shinya ; Mabuchi, IsseiCalyculin-A (CLA), a protein phosphatase inhibitor, has been known to induce cleavage resembling normal furrowing in unfertilized sea urchin eggs. In CLA-treated eggs, actin filaments and myosin assemble to form a contractile ring-like structure in the egg cortex; however, this occurs in the absence of a mitotic spindle or asters. Here, we investigated the relationship between the plane of CLA-induced cleavage and the intrinsic animal-vegetal polar axis in sea urchin eggs. The animal-vegetal axis was established using black ink to visualize the jelly canal located at the animal pole in the jelly coat surrounding the egg. We measured the acute angle between the jelly canal axis and the cleavage plane for both fertilized eggs and CLA-treated unfertilized eggs. Although the acute angle lay within 10 degrees for most of the fertilized eggs, it varied widely for CLA-treated unfertilized eggs. Measurements of the diameter of blastomeres revealed that cleavage of fertilized eggs took place in the mid-plane of the egg, but that CLA-induced divisions were unequal. These results suggest that neither the orientation nor the location of the CLA-induced cleavage furrow is related to the animal-vegetal polar axis of the egg, even though the furrowing mechanism itself is not dissimilar to that in fertilized eggs.
-
ArticleMammalian end binding proteins control persistent microtubule growth(Rockefeller University Press, 2009-03-02) Komarova, Yulia A. ; De Groot, Christian O. ; Grigoriev, Ilya ; Gouveia, Susana Montenegro ; Munteanu, E. Laura ; Schober, Joseph M. ; Honnappa, Srinivas ; Buey, Ruben M. ; Hoogenraad, Casper C. ; Dogterom, Marileen ; Borisy, Gary G. ; Steinmetz, Michel O. ; Akhmanova, AnnaEnd binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.
-
PreprintMigration and actin protrusion in melanoma cells are regulated by EB1 protein( 2009-04) Schober, Joseph M. ; Cain, Jeanine M. ; Komarova, Yulia A. ; Borisy, Gary G.Remodeling of actin and microtubule cytoskeletons is thought to be coupled; however, the interplay between these two systems is not fully understood. We show a microtubule end-binding protein, EB1, is required for formation of polarized morphology and motility of melanoma cells. EB1 depletion decreased lamellipodia protrusion, and resulted in loss of opposed protruding and retracting cell edges. Lamellipodia attenuation correlated with mis-localization of filopodia throughout the cell and decreased Arp3 localization. EB1-depleted cells displayed less persistent migration and reduced velocity in singlecell motility experiments. We propose EB1 coordinates melanoma cell migration through regulating the balance between lamellipodial and filopodial protrusion.