Publications

* joined first author # joined corresponding author

2022
Benjamin Dalton✳︎, David Oriola✳︎, Franziska Decker✳︎, Frank Jülicher#, Jan Brugués#
A gelation transition enables the self-organization of bipolar metaphase spindles.
Nat Phys, 18(3) 323-331 (2022)
Open Access DOI
The mitotic spindle is a highly dynamic bipolar structure that emerges from the self-organization of microtubules, molecular motors and other proteins. Sustained motor-driven poleward flows of dynamic microtubules play a key role in the bipolar organization of spindles. However, it is not understood how the local activity of motor proteins generates these large-scale coherent poleward flows. Here we show that a gelation transition enables long-range microtubule transport causing the spindles to self-organize into two oppositely polarized microtubule gels. Laser ablation experiments reveal that local active stresses generated at the spindle midplane propagate through the structure, thereby driving global coherent microtubule flows. Simulations show that microtubule gels undergoing rapid turnover can exhibit long stress relaxation times, in agreement with the long-range flows observed in experiments. Finally, our model predicts that in the presence of branching microtubule nucleation, either disrupting such flows or decreasing the network connectivity can lead to a microtubule polarity reversal in spindles. We experimentally confirm this inversion of polarity by abolishing the microtubule transport in spindles. Overall, we uncover a connection between spindle rheology and architecture in spindle self-organization. The activity of molecular motors drives the self-organization of cytoskeleton structures, leading to large-scale active flows. Now, experiments and simulations show how a gelation process enables such long-range transport in spindles.


Jose A. Morin✳︎, Sina Wittmann✳︎, Sandeep Choubey✳︎, Adam Klosin, Stefan Golfier, Anthony A. Hyman#, Frank Jülicher#, Stephan W. Grill#
Sequence-dependent surface condensation of a pioneer transcription factor on DNA.
Nat Phys, 18(3) 271-276 (2022)
Open Access DOI
Biomolecular condensates are dense assemblies of proteins that form distinct biochemical compartments without being surrounded by a membrane. Some, such as P granules and stress granules, behave as droplets and contain many millions of molecules. Others, such as transcriptional condensates that form on the surface of DNA, are small and contain thousands of molecules. The physics behind the formation of small condensates on DNA surfaces is still under discussion. Here we investigate the nature of transcription factor condensates using the pioneer transcription factor Kruppel-like factor 4 (Klf4). We show that Klf4 can phase separate on its own at high concentrations, but at low concentrations, Klf4 only forms condensates on DNA. Using optical tweezers, we demonstrate that these Klf4 condensates form on DNA as a type of surface condensation. This surface condensation involves a switch-like transition from a thin adsorbed layer to a thick condensed layer, which shows hallmarks of a prewetting transition. The localization of condensates on DNA correlates with sequence, suggesting that the condensate formation of Klf4 on DNA is a sequence-dependent form of surface condensation. Prewetting together with sequence specificity can explain the size and position control of surface condensates. We speculate that a prewetting transition of pioneer transcription factors on DNA underlies the formation and positioning of transcriptional condensates and provides robustness to transcriptional regulation. A DNA-binding protein condenses on DNA via a switch-like transition. Surface condensation occurs at preferential DNA locations suggesting collective sequence readout and enabling sequence-specificity robustness with respect to protein concentration.


2021
Thomas Quail, Stefan Golfier, Maria Elsner, Keisuke Ishihara, Vasanthanarayan Murugesan, Roman Renger, Frank Jülicher#, Jan Brugués#
Force generation by protein-DNA co-condensation.
Nat Phys, 17(9) 1007-1012 (2021)
Open Access DOI
Interactions between liquids and surfaces generate forces(1,2) that are crucial for many processes in biology, physics and engineering, including the motion of insects on the surface of water(3), modulation of the material properties of spider silk(4) and self-assembly of microstructures(5). Recent studies have shown that cells assemble biomolecular condensates via phase separation(6). In the nucleus, these condensates are thought to drive transcription(7), heterochromatin formation(8), nucleolus assembly(9) and DNA repair(10). Here we show that the interaction between liquid-like condensates and DNA generates forces that might play a role in bringing distant regulatory elements of DNA together, a key step in transcriptional regulation. We combine quantitative microscopy, in vitro reconstitution, optical tweezers and theory to show that the transcription factor FoxA1 mediates the condensation of a protein-DNA phase via a mesoscopic first-order phase transition. After nucleation, co-condensation forces drive growth of this phase by pulling non-condensed DNA. Altering the tension on the DNA strand enlarges or dissolves the condensates, revealing their mechanosensitive nature. These findings show that DNA condensation mediated by transcription factors could bring distant regions of DNA into close proximity, suggesting that this physical mechanism is a possible general regulatory principle for chromatin organization that may be relevant in vivo.


Chang-Yu Chang✳︎, Jean C C Vila✳︎, Madeline Bender, Richard Li, Madeleine C Mankowski, Molly Bassette, Julia Borden, Stefan Golfier, Paul Gerald L Sanchez, Rachel Waymack, Xinwen Zhu, Juan Diaz-Colunga, Sylvie Estrela, Maria Rebolleda-Gomez, Alvaro Sanchez
Engineering complex communities by directed evolution.
Nat Ecol Evol, 5(7) 1011-1023 (2021)
DOI
Directed evolution has been used for decades to engineer biological systems at or below the organismal level. Above the organismal level, a small number of studies have attempted to artificially select microbial ecosystems, with uneven and generally modest success. Our theoretical understanding of artificial ecosystem selection is limited, particularly for large assemblages of asexual organisms, and we know little about designing efficient methods to direct their evolution. Here, we have developed a flexible modelling framework that allows us to systematically probe any arbitrary selection strategy on any arbitrary set of communities and selected functions. By artificially selecting hundreds of in silico microbial metacommunities under identical conditions, we first show that the main breeding methods used to date, which do not necessarily let communities reach their ecological equilibrium, are outperformed by a simple screen of sufficiently mature communities. We then identify a range of alternative directed evolution strategies that, particularly when applied in combination, are well suited for the top-down engineering of large, diverse and stable microbial consortia. Our results emphasize that directed evolution allows an ecological structure-function landscape to be navigated in search of dynamically stable and ecologically resilient communities with desired quantitative attributes.


Benjamin Dalton✳︎, David Oriola✳︎, Franziska Decker✳︎, Frank Jülicher, Jan Brugués
A gelation transition enables the self-organization of bipolar metaphase spindles
bioRxiv, Art. No. https://doi.org/10.1101/2021.01.15.426844 (2021)
Open Access DOI

Keisuke Ishihara, Franziska Decker, Paulo Caldas, James F. Pelletier, Martin Loose, Jan Brugués, Timothy J. Mitchison
Spatial Variation of Microtubule Depolymerization in Large Asters.
Mol Biol Cell, 32(9) 869-879 (2021)
DOI
Microtubule plus end depolymerization rate is a potentially important target of physiological regulation, but it has been challenging to measure, so its role in spatial organization is poorly understood. Here we apply a method for tracking plus ends based on time difference imaging to measure depolymerization rates in large interphase asters growing in Xenopus egg extract. We observed strong spatial regulation of depolymerization rates, which were higher in the aster interior compared to the periphery, and much less regulation of polymerization or catastrophe rates. We interpret these data in terms of a limiting component model, where aster growth results in lower levels of soluble tubulin and MAPs in the interior cytosol compared to that at the periphery. The steady-state polymer fraction of tubulin was ∼30%, so tubulin is not strongly depleted in the aster interior. We propose that the limiting component for microtubule assembly is a MAP that inhibits depolymerization, and that egg asters are tuned to low microtubule density. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].


Keisuke Ishihara✳︎, Arghyadip Mukherjee✳︎, Elena Gromberg, Jan Brugués#, Elly M. Tanaka#, Frank Jülicher#
Topological morphogenesis of neuroepithelial organoids.
bioRxiv, Art. No. https://doi.org/10.1101/2021.08.08.455385 (2021)
Open Access DOI
Animal organs exhibit complex topologies involving cavities and tubular networks, which underlie their form and function. However, how topology emerges during organ morphogenesis remains elusive. Here, we combine tissue reconstitution and quantitative microscopy to show that trans and cis epithelial fusion govern tissue topology and shape. These two modes of topological transitions can be regulated in neuroepithelial organoids, leading to divergent topologies. The morphological space can be captured by a single control parameter which is analogous to the reduced Gaussian rigidity of an epithelial surface. Finally, we identify a pharmacologically accessible pathway that regulates the frequency of trans and cis fusion, and demonstrate the control of organoid topology and shape. The physical principles uncovered here provide fundamental insights into the self-organization of complex tissues.


2020
Elisa Maria Rieckhoff, Frederic Berndt, Maria Elsner, Stefan Golfier, Franziska Decker, Keisuke Ishihara, Jan Brugués
Spindle Scaling Is Governed by Cell Boundary Regulation of Microtubule Nucleation.
Curr Biol, 30(24) 4973-4983 (2020)
Open Access DOI
Cellular organelles such as the mitotic spindle adjust their size to the dimensions of the cell. It is widely understood that spindle scaling is governed by regulation of microtubule polymerization. Here, we use quantitative microscopy in living zebrafish embryos and Xenopus egg extracts in combination with theory to show that microtubule polymerization dynamics are insufficient to scale spindles and only contribute below a critical cell size. In contrast, microtubule nucleation governs spindle scaling for all cell sizes. We show that this hierarchical regulation arises from the partitioning of a nucleation inhibitor to the cell membrane. Our results reveal that cells differentially regulate microtubule number and length using distinct geometric cues to maintain a functional spindle architecture over a large range of cell sizes.


Patrick M McCall, Kyoohyun Kim, Anatol W Fritsch, Juan M Iglesias-Artola, Louise Jawerth, Jie Wang, Martine Ruer, Jan Peychl, Andrey Poznyakovskiy, Jochen Guck, Simon Alberti, Anthony A. Hyman, Jan Brugues
Quantitative phase microscopy enables precise and efficient determination of biomolecular condensate composition.
bioRxiv, Art. No. https://doi.org/10.1101/2020.10.25.352823 (2020)
Open Access DOI
Many compartments in eukaryotic cells are protein-rich biomolecular condensates demixed from the cyto- or nucleoplasm. Although much has been learned in recent years about the integral roles condensates play in many cellular processes as well as the biophysical properties of reconstituted condensates, an understanding of their most basic feature, their composition, remains elusive. Here we combined quantitative phase microscopy (QPM) and the physics of sessile droplets to develop a precise method to measure the shape and composition of individual model condensates. This technique does not rely on fluorescent dyes or tags, which we show can significantly alter protein phase behavior, and requires 1000-fold less material than traditional label-free technologies. We further show that this QPM method measures the protein concentration in condensates to a 3-fold higher precision than the next best label-free approach, and that commonly employed strategies based on fluorescence intensity dramatically underestimate these concentrations by as much as 50-fold. Interestingly, we find that condensed-phase protein concentrations can span a broad range, with PGL3, TAF15(RBD) and FUS condensates falling between 80 and 500 mg/ml under typical in vitro conditions. This points to a natural diversity in condensate composition specified by protein sequence. We were also able to measure temperature-dependent phase equilibria with QPM, an essential step towards relating phase behavior to the underlying physics and chemistry. Finally, time-resolved QPM reveals that PGL3 condensates undergo a contraction-like process during aging which leads to doubling of the internal protein concentration coupled to condensate shrinkage. We anticipate that this new approach will enable understanding the physical properties of biomolecular condensates and their function.


Thomas Quail, Stefan Golfier, Maria Elsner, Keisuke Ishihara, Frank Jülicher, Jan Brugués
Capillary forces drive pioneer transcription factor-mediated DNA condensation.
bioRxiv, Art. No. https://doi.org/10.1101/2020.09.17.302299 (2020)
Open Access DOI
Capillary forces are driven by interactions between liquids and surfaces1. These forces are crucial for many processes ranging from biology and physics to engineering, such as the motion of aquatic insects on the surface of water2, modulation of the material properties of spider silk3,4, and self-assembly of small objects and microstructures5. Recent studies have shown that cells assemble biomolecular condensates in a manner similar to phase separation6. In the nucleus, these condensates are thought to drive transcription7–10, heterochromatin formation11,12, nucleolus assembly13,14, and DNA repair15,16. Here, we test if the interaction between liquid-like condensates and chromatin generates capillary forces, which might play a role in bringing distant regulatory elements of DNA together, a key step in transcriptional regulation. We combine quantitative microscopy, in vitro reconstitution, and theory to show that the pioneer transcription factor FoxA1 mediates the condensation of a DNA-protein phase via a mesoscopic first-order phase transition. Surprisingly, after nucleation, capillary forces drive growth of this phase by pulling non-condensed DNA. Altering the tension on the DNA strand enlarges or dissolves the condensates, revealing their mechanosensitive nature. These findings show that DNA condensation mediated by transcription factors could bring distant regions of DNA in close proximity using capillary forces. Our work suggests that the physics of DNA and protein condensation driven by capillary forces provides a general regulatory principle for chromatin organization.


Keisuke Ishihara#, Ashish B George, Ryan Cornelius, Kirill S Korolev#
Traveling fronts in self-replicating persistent random walks with multiple internal states.
New J Phys, 22(8) Art. No. 083034 (2020)
Open Access DOI
Self-activation coupled to a transport mechanism results in traveling waves that describe polymerization reactions, forest fires, tumor growth, and even the spread of epidemics. Diffusion is a simple and commonly used model of particle transport. Many physical and biological systems are, however, better described by persistent random walks that switch between multiple states of ballistic motion. So far, traveling fronts in persistent random walk models have only been analyzed in special, simplified cases. Here, we formulate the general model of reaction-transport processes in such systems and show how to compute the expansion velocity for arbitrary number of states. For the two-state model, we obtain a closed-form expression for the velocity and report how it is affected by different transport and replication parameters. We also show that nonzero death rates result in a discontinuous transition from quiescence to propagation. We compare our results to a recent observation of a discontinuous onset of propagation in microtubule asters and comment on the universal nature of the underlying mechanism.


David Oriola, Frank Jülicher#, Jan Brugués#
Active forces shape the metaphase spindle through a mechanical instability.
Proc Natl Acad Sci U.S.A., 117(28) 16154-16159 (2020)
DOI
The metaphase spindle is a dynamic structure orchestrating chromosome segregation during cell division. Recently, soft matter approaches have shown that the spindle behaves as an active liquid crystal. Still, it remains unclear how active force generation contributes to its characteristic spindle-like shape. Here we combine theory and experiments to show that molecular motor-driven forces shape the structure through a barreling-type instability. We test our physical model by titrating dynein activity in Xenopus egg extract spindles and quantifying the shape and microtubule orientation. We conclude that spindles are shaped by the interplay between surface tension, nematic elasticity, and motor-driven active forces. Our study reveals how motor proteins can mold liquid crystalline droplets and has implications for the design of active soft materials.


Keisuke Ishihara, Franziska Decker, Paulo Caldas, James F. Pelletier, Martin Loose, Jan Brugués, Timothy J. Mitchison
Spatial Variation of Microtubule Depolymerization in Large Asters Suggests Regulation by MAP Depletion.
bioRxiv, Art. No. https://doi.org/10.1101/2020.06.26.172783 (2020)
Open Access DOI
Microtubule plus end depolymerization rate is a potentially important target of physiological regulation, but it has been challenging to measure, so its role in spatial organization is poorly understood. Here we apply a method for tracking plus ends based on time difference imaging to measure depolymerization rates in large interphase asters growing in Xenopus egg extract. We observed strong spatial regulation of depolymerization rates, which were almost two-fold higher in the aster interior compared to the periphery, and much less regulation of polymerization or catastrophe rates. We interpret these data in terms of a limiting component model, where aster growth results in lower levels of soluble tubulin and MAPs in the interior cytosol compared to that at the periphery. The steady-state polymer fraction of tubulin was ~30%, so tubulin is not strongly depleted in the aster interior. We propose that the limiting component for microtubule assembly is a MAP that inhibits depolymerization, and that egg asters are tuned to low microtubule density.


Elisa Maria Rieckhoff, Frederic Berndt, Stefan Golfier, Franziska Decker, Maria Elsner, Keisuke Ishihara, Jan Brugués
Spindle scaling is governed by cell boundary regulation of microtubule nucleation.
bioRxiv, Art. No. https://doi.org/10.1101/2020.06.15.136937 (2020)
Open Access DOI
Cellular organellessuch as the mitotic spindleadjust their size to the dimensions of the cell. It is widely understood that spindle scaling is governed by regulation of microtubule polymerization.Here weusequantitative microscopyin living zebrafish embryos andXenopuseggextractsin combination with theoryto show that microtubule polymerization dynamics are insufficient to scale spindles and only contribute below a critical cell size. In contrast, microtubule nucleation governs spindle scaling for all cell sizes. We show that this hierarchical regulation arisesfrom the partitioning of a nucleation inhibitor to the cell membrane. Our results reveal that cells differentially regulatemicrotubule number and length using distinct geometric cues to maintain a functional spindle architecture over a large range of cell sizes.


Stefan Golfier, Thomas Quail, Hiroshi Kimura, Jan Brugués
Cohesin and condensin extrude DNA loops in a cell-cycle dependent manner.
Elife, 9 Art. No. e53885 (2020)
Open Access DOI
Loop extrusion by structural maintenance of chromosomes complexes (SMCs) has been proposed as a mechanism to organize chromatin in interphase and metaphase. However, the requirements for chromatin organization in these cell cycle phases are different, and it is unknown whether loop extrusion dynamics and the complexes that extrude DNA also differ. Here, we used Xenopus egg extracts to reconstitute and image loop extrusion of single DNA molecules during the cell cycle. We show that loops form in both metaphase and interphase, but with distinct dynamic properties. Condensin extrudes DNA loops non-symmetrically in metaphase, whereas cohesin extrudes loops symmetrically in interphase. Our data show that loop extrusion is a general mechanism underlying DNA organization, with dynamic and structural properties that are biochemically regulated during the cell cycle.


Jonathan Rodenfels, Pablo Sartori, Stefan Golfier, Kartikeya Nagendra, Karla M. Neugebauer, Jonathon Howard
Contribution of increasing plasma membrane to the energetic cost of early zebrafish embryogenesis.
Mol Biol Cell, 31(7) 520-526 (2020)
DOI
How do early embryos allocate the resources stored in the sperm and egg? Recently, we established isothermal calorimetry to measure heat dissipation by living zebra-fish embryos and to estimate the energetics of specific developmental events. During the reductive cleavage divisions, the rate of heat dissipation increases from ∼60 nJ · s-1 at the two-cell stage to ∼90 nJ · s-1 at the 1024-cell stage. Here we ask which cellular process(es) drive this increasing energetic cost. We present evidence that the cost is due to the increase in the total surface area of all the cells of the embryo. First, embryo volume stays constant during the cleavage stage, indicating that the increase is not due to growth. Second, the heat increase is blocked by nocodazole, which inhibits DNA replication, mitosis, and cell division; this suggests some aspect of cell proliferation contributes to these costs. Third, the heat increases in proportion to the total cell surface area rather than total cell number. Fourth, the heat increase falls within the range of the estimated costs of maintaining and assembling plasma membranes and associated proteins. Thus, the increase in total plasma membrane associated with cell proliferation is likely to contribute appreciably to the total energy budget of the embryo.


Gunar Fabig#, Robert Kiewisz, Norbert Lindow, James A Powers, Vanessa Cota, Luis J Quintanilla, Jan Brugués, Steffen Prohaska, Diana S Chu, Thomas Müller-Reichert#
Male meiotic spindle features that efficiently segregate paired and lagging chromosomes.
Elife, 9 Art. No. e50988 (2020)
Open Access DOI
Chromosome segregation during male meiosis is tailored to rapidly generate multitudes of sperm. Little is known about mechanisms that efficiently partition chromosomes to produce sperm. Using live imaging and tomographic reconstructions of spermatocyte meiotic spindles in Caenorhabditis elegans, we find the lagging X chromosome, a distinctive feature of anaphase I in C. elegans males, is due to lack of chromosome pairing. The unpaired chromosome remains tethered to centrosomes by lengthening kinetochore microtubules, which are under tension, suggesting that a 'tug of war' reliably resolves lagging. We find spermatocytes exhibit simultaneous pole-to-chromosome shortening (anaphase A) and pole-to-pole elongation (anaphase B). Electron tomography unexpectedly revealed spermatocyte anaphase A does not stem solely from kinetochore microtubule shortening. Instead, movement of autosomes is largely driven by distance change between chromosomes, microtubules, and centrosomes upon tension release during anaphase. Overall, we define novel features that segregate both lagging and paired chromosomes for optimal sperm production.


David Oriola, Frank Jülicher, Jan Brugués
Active force generation shapes the metaphase spindle through a mechanical instability.
bioRxiv, Art. No. https://doi.org/10.1101/2020.02.08.939868 (2020)
Open Access DOI
The metaphase spindle is a dynamic structure that segregates chromosomes during cell division. Recently, soft matter approaches have shown that the spindle behaves as an active liquid crystal. Still, it remains unclear how active force generation contributes to its characteristic spindle-like shape. Here, we combine theory and experiments to show that molecular motor driven forces shape the structure through a barreling-type instability. We test our physical model by titrating dynein activity in Xenopus egg extract spindles and quantifying the shape and microtubule orientation. We conclude that spindles are shaped by the interplay between surface tension, nematic elasticity and motor-driven active forces. Our study reveals how active force generation can mold liquid crystal droplets and it has implications on the morphology of non-membrane bound compartments demixed from the cytoplasm.


2019
Paulo Caldas, Mar López-Pelegrín, Daniel J G Pearce, Nazmi Burak Budanur, Jan Brugués, Martin Loose
Cooperative ordering of treadmilling filaments in cytoskeletal networks of FtsZ and its crosslinker ZapA.
Nat Commun, 10(1) Art. No. 5744 (2019)
Open Access DOI
During bacterial cell division, the tubulin-homolog FtsZ forms a ring-like structure at the center of the cell. This Z-ring not only organizes the division machinery, but treadmilling of FtsZ filaments was also found to play a key role in distributing proteins at the division site. What regulates the architecture, dynamics and stability of the Z-ring is currently unknown, but FtsZ-associated proteins are known to play an important role. Here, using an in vitro reconstitution approach, we studied how the well-conserved protein ZapA affects FtsZ treadmilling and filament organization into large-scale patterns. Using high-resolution fluorescence microscopy and quantitative image analysis, we found that ZapA cooperatively increases the spatial order of the filament network, but binds only transiently to FtsZ filaments and has no effect on filament length and treadmilling velocity. Together, our data provides a model for how FtsZ-associated proteins can increase the precision and stability of the bacterial cell division machinery in a switch-like manner.


Johannes Baumgart✳︎, Marcel Kirchner✳︎, Stefanie Redemann, Alec Bond, Jeffrey Woodruff, Jean-Marc Verbavatz, Frank Jülicher, Thomas Müller-Reichert, Anthony Hyman, Jan Brugués
Soluble tubulin is significantly enriched at mitotic centrosomes.
J Cell Biol, 218(12) 3977-3985 (2019)
DOI
During mitosis, the centrosome expands its capacity to nucleate microtubules. Understanding the mechanisms of centrosomal microtubule nucleation is, however, constrained by a lack of knowledge of the amount of soluble and polymeric tubulin at mitotic centrosomes. Here we combined light microscopy and serial-section electron tomography to measure the amount of dimeric and polymeric tubulin at mitotic centrosomes in early C. elegans embryos. We show that a C. elegans one-cell stage centrosome at metaphase contains >10,000 microtubules with a total polymer concentration of 230 µM. Centrosomes concentrate soluble α/β tubulin by about 10-fold over the cytoplasm, reaching peak values of 470 µM, giving a combined total monomer and polymer tubulin concentration at centrosomes of up to 660 µM. These findings support in vitro data suggesting that microtubule nucleation in C. elegans centrosomes is driven in part by concentrating soluble tubulin.


Stefan Golfier, Thomas Quail, Hiroshi Kimura, Jan Brugués
Cohesin and condensin extrude loops in a cell-cycle dependent manner
bioRxiv, Art. No. https://doi.org/10.1101/821306 (2019)
Open Access DOI
Chromatin undergoes a dramatic reorganization during the cell cycle1–3. In interphase, chromatin is organized into compartments and topological-associating domains (TADs) that are cell-type specific4–7, whereas in metaphase, chromosomes undergo large-scale compaction, leading to the loss of specific boundaries and the shutdown of transcription8–12. Loop extrusion by structural maintenance of chromosomes complexes (SMCs) has been proposed as a mechanism to organize chromatin in interphase and metaphase13–19. However, the requirements for chromatin organization in these cell phases are very different, and it is unknown whether loop extrusion dynamics and the complexes that extrude them also differ. Here, we used Xenopus egg extracts to reconstitute and image loop extrusion of single DNA molecules during the cell cycle. We show that loops form in both metaphase and interphase, but with distinct dynamic properties. Condensin extrudes asymmetric loops in metaphase, whereas cohesin extrudes symmetric loops in interphase. Our data show that loop extrusion is a general mechanism for the organization of DNA, with dynamic and structural properties that are molecularly regulated during the cell cycle.


Elisa Maria Rieckhoff, Keisuke Ishihara, Jan Brugués
How to tune spindle size relative to cell size?
Curr Opin Cell Biol, 60 139-144 (2019)
DOI
Cells need to regulate the size and shape of their organelles for proper function. For example, the mitotic spindle adapts its size to changes in cell size over several orders of magnitude, but we lack a mechanistic understanding of how this is achieved. Here, we review our current knowledge of how small and large spindles assemble and ask which microtubule-based biophysical processes (nucleation, polymerization dynamics, transport) may be responsible for spindle size regulation. Finally, we review possible cell-scale mechanisms that put spindle size under the regulation of cell size.


2018
Frederic Berndt, Gopi Shah, Rory M Power, Jan Brugués, Jan Huisken
Dynamic and non-contact 3D sample rotation for microscopy.
Nat Commun, 9(1) Art. No. 5025 (2018)
Open Access DOI
Precise sample orientation is crucial for microscopy but is often performed with macroscopic tools and low accuracy. In vivo imaging of growing and developing samples even requires dynamic adaptation of the sample orientation to continuously achieve optimal imaging. Here, we present a method for freely positioning a sample in 3D by introducing magnetic beads and applying a magnetic field. We demonstrate magnetic orientation of fixed mouse embryos and artemia, and live zebrafish embryos and larvae on an epi-fluorescence microscope and on a light-sheet system for optimal imaging.


David Oriola, Daniel Needleman, Jan Brugués
The Physics of the Metaphase Spindle.
Ann Rev Biophys, 47 655-673 (2018)
DOI
The assembly of the mitotic spindle and the subsequent segregation of sister chromatids are based on the self-organized action of microtubule filaments, motor proteins, and other microtubule-associated proteins, which constitute the fundamental force-generating elements in the system. Many of the components in the spindle have been identified, but until recently it remained unclear how their collective behaviors resulted in such a robust bipolar structure. Here, we review the current understanding of the physics of the metaphase spindle that is only now starting to emerge.


Franziska Decker, David Oriola, Benjamin Dalton, Jan Brugues
Autocatalytic microtubule nucleation determines the size and mass of Xenopus laevis egg extract spindles.
Elife, 7 Art. No. e31149 (2018)
Open Access DOI
Regulation of size and growth is a fundamental problem in biology. A prominent example is the formation of the mitotic spindle, where protein concentration gradients around chromosomes are thought to regulate spindle growth by controlling microtubule nucleation. Previous evidence suggests that microtubules nucleate throughout the spindle structure. However, the mechanisms underlying microtubule nucleation and its spatial regulation are still unclear. Here, we developed an assay based on laser ablation to directly probe microtubule nucleation events in Xenopuslaevis egg extracts. Combining this method with theory and quantitative microscopy, we show that the size of a spindle is controlled by autocatalytic growth of microtubules, driven by microtubule-stimulated microtubule nucleation. The autocatalytic activity of this nucleation system is spatially regulated by the limiting amounts of active microtubule nucleators, which decrease with distance from the chromosomes. This mechanism provides an upper limit to spindle size even when resources are not limiting.


2017
Stefanie Redemann, Johannes Baumgart, Norbert Lindow, Michael Shelley#, Ehssan Nazockdast, Andrea Kratz, Steffen Prohaska, Jan Brugués, Sebastian Fürthauer, Thomas Müller-Reichert#
C. elegans chromosomes connect to centrosomes by anchoring into the spindle network.
Nat Commun, 8 Art. No. 15288 (2017)
Open Access DOI
The mitotic spindle ensures the faithful segregation of chromosomes. Here we combine the first large-scale serial electron tomography of whole mitotic spindles in early C. elegans embryos with live-cell imaging to reconstruct all microtubules in 3D and identify their plus- and minus-ends. We classify them as kinetochore (KMTs), spindle (SMTs) or astral microtubules (AMTs) according to their positions, and quantify distinct properties of each class. While our light microscopy and mutant studies show that microtubules are nucleated from the centrosomes, we find only a few KMTs directly connected to the centrosomes. Indeed, by quantitatively analysing several models of microtubule growth, we conclude that minus-ends of KMTs have selectively detached and depolymerized from the centrosome. In toto, our results show that the connection between centrosomes and chromosomes is mediated by an anchoring into the entire spindle network and that any direct connections through KMTs are few and likely very transient.


Jan Brugués
Cytoskeleton Dynamics: Mind the Gap!
Curr Biol, 27(7) 279-281 (2017)
DOI
A new study presents a quantitative biophysical model of microtubule aster growth with autocatalytic microtubule nucleation. The model accounts for asters that grow indefinitely, even when their microtubules are unstable.


2015
Ricard Alert, Jaume Casademunt, Jan Brugués, Pierre Sens
Model for probing membrane-cortex adhesion by micropipette aspiration and fluctuation spectroscopy.
Biophys J, 108(8) 1878-1886 (2015)
DOI
We propose a model for membrane-cortex adhesion that couples membrane deformations, hydrodynamics, and kinetics of membrane-cortex ligands. In its simplest form, the model gives explicit predictions for the critical pressure for membrane detachment and for the value of adhesion energy. We show that these quantities exhibit a significant dependence on the active acto-myosin stresses. The model provides a simple framework to access quantitative information on cortical activity by means of micropipette experiments. We also extend the model to incorporate fluctuations and show that detailed information on the stability of membrane-cortex coupling can be obtained by a combination of micropipette aspiration and fluctuation spectroscopy measurements.


Franziska Decker, Jan Brugués
Dissecting microtubule structures by laser ablation.
Methods Cell Biol, 125 61-75 (2015)
DOI
Here, we describe a detailed protocol, based on laser ablation and fluorescence optical microscopy, to measure the microtubule organization in spindles, including microtubule length distribution, polarity, and plus and minus end densities. The method uses the asymmetry in microtubule depolymerization after a cut, where the newly created microtubule plus ends depolymerize all the way to the minus ends, whereas the newly created minus ends remain stable. The protocol described in this chapter is optimized for spindles, but can be easily applied to any microtubule-based structure. The chapter is divided into two parts. First, we provide the theoretical basis for the method. Second, we describe in detail all steps necessary to reconstruct the microtubule organization of a spindle assembled in Xenopus laevis egg extract. Compared to electron microscopy, which in theory can resolve individual microtubules in spindles and provide similar structural information, our method is fast and simple enough to allow for a full quantitative reconstruction of the microtubule organization of several X. laevis spindles—which have volumes tens of thousands of times larger than spindles whose structures have been previously solved by electron microscopy—in a single experimental session, as well as to explore how the architecture of these structures changes in response to biochemical perturbations.


2014
Jan Brugues, Daniel Needleman
Physical basis of spindle self-organization.
Proc Natl Acad Sci U.S.A., 111(52) 18496-18500 (2014)
PDF DOI
The cytoskeleton forms a variety of steady-state, subcellular structures that are maintained by continuous fluxes of molecules and energy. Understanding such self-organizing structures is not only crucial for cell biology but also poses a fundamental challenge for physics, since these systems are active materials that behave drastically differently from matter at or near equilibrium. Active liquid crystal theories have been developed to study the self-organization of cytoskeletal filaments in in vitro systems of purified components. However, it has been unclear how relevant these simplified approaches are for understanding biological structures, which can be composed of hundreds of distinct proteins. Here we show that a suitably constructed active liquid crystal theory produces remarkably accurate predictions of the behaviors of metaphase spindles-the cytoskeletal structure, composed largely of microtubules and associated proteins, that segregates chromosomes during cell division.


Che-Hang Yu, Noah Langowitz, Hai-Yin Wu, Reza Farhadifar, Jan Brugues, Tae Yeon Yoo, Daniel Needleman
Measuring microtubule polarity in spindles with second-harmonic generation.
Biophys J, 106(8) 1578-1587 (2014)
PDF DOI
The spatial organization of microtubule polarity, and the interplay between microtubule polarity and protein localization, is thought to be crucial for spindle assembly, anaphase, and cytokinesis, but these phenomena remain poorly understood, in part due to the difficulty of measuring microtubule polarity in spindles. We develop and implement a method to nonperturbatively and quantitatively measure microtubule polarity throughout spindles using a combination of second-harmonic generation and two-photon fluorescence. We validate this method using computer simulations and by comparison to structural data on spindles obtained from electron tomography and laser ablation. This method should provide a powerful tool for studying spindle organization and function, and may be applicable for investigating microtubule polarity in other systems.


Dan Needleman, Jan Brugues
Determining physical principles of subcellular organization.
Dev Cell, 29(2) 135-138 (2014)
DOI
Recent advances have transformed our understanding of cell biology, but we are still unable to predict the behaviors of these systems. One difficulty is that we lack an understanding of the physical principles of subcellular organization. Combining quantitative experiments with new theoretical insights may allow such principles to be developed.


2012
Jan Brugués, Valeria Nuzzo, Eric Mazur, Daniel Needleman
Nucleation and transport organize microtubules in metaphase spindles.
Cell, 149(3) 554-564 (2012)
DOI
Spindles are arrays of microtubules that segregate chromosomes during cell division. It has been difficult to validate models of spindle assembly due to a lack of information on the organization of microtubules in these structures. Here we present a method, based on femtosecond laser ablation, capable of measuring the detailed architecture of spindles. We used this method to study the metaphase spindle in Xenopus laevis egg extracts and found that microtubules are shortest near poles and become progressively longer toward the center of the spindle. These data, in combination with mathematical modeling, imaging, and biochemical perturbations, are sufficient to reject previously proposed mechanisms of spindle assembly. Our results support a model of spindle assembly in which microtubule polymerization dynamics are not spatially regulated, and the proper organization of microtubules in the spindle is determined by nonuniform microtubule nucleation and the local sorting of microtubules by transport.


2010
Javier G Orlandi, Carles Blanch-Mercader, Jan Brugués, Jaume Casademunt
Cooperativity of self-organized Brownian motors pulling on soft cargoes.
Phys Rev E, 82(6 Pt 1) 61903-61903 (2010)
DOI
We study the cooperative dynamics of Brownian motors moving along a one-dimensional track when an external load is applied to the leading motor, mimicking molecular motors pulling on membrane-bound cargoes in intracellular traffic. Due to the asymmetric loading, self-organized motor clusters form spontaneously. We model the motors with a two-state noise-driven ratchet formulation and study analytically and numerically the collective velocity-force and efficiency-force curves resulting from mutual interactions, mostly hard-core repulsion and weak (nonbinding) attraction. We analyze different parameter regimes including the limits of weak noise, mean-field behavior, rigid coupling, and large numbers of motors, for the different interactions. We present a general framework to classify and quantify cooperativity. We show that asymmetric loading leads generically to enhanced cooperativity beyond the simple superposition of the effects of individual motors. For weakly attracting interactions, the cooperativity is mostly enhanced, including highly coordinated motion of motors and complex nonmonotonic velocity-force curves, leading to self-regulated clusters. The dynamical scenario is enriched by resonances associated to commensurability of different length scales. Large clusters exhibit synchronized dynamics and bidirectional motion. Biological implications are discussed.


Benoît Maugis, Jan Brugués, Pierre Nassoy, Nancy Guillen, Pierre Sens, François Amblard
Dynamic instability of the intracellular pressure drives bleb-based motility.
J Cell Sci, 123(Pt 22) 3884-3892 (2010)
DOI
We have demonstrated that the two- and three-dimensional motility of the human pathogenic parasite Entamoeba histolytica (Eh) depends on sustained instability of the intracellular hydrostatic pressure. This instability drives the cyclic generation and healing of membrane blebs, with typical protrusion velocities of 10-20 μm/second over a few hundred milliseconds and healing times of 10 seconds. The use of a novel micro-electroporation method to control the intracellular pressure enabled us to develop a qualitative model with three parameters: the rate of the myosin-driven internal pressure increase; the critical disjunction stress of membrane-cytoskeleton bonds; and the turnover time of the F-actin cortex. Although blebs occur randomly in space and irregularly time, they can be forced to occur with a defined periodicity in confined geometries, thus confirming our model. Given the highly efficient bleb-based motility of Eh in vitro and in vivo, Eh cells represent a unique model for studying the physical and biological aspects of amoeboid versus mesenchymal motility in two- and three-dimensional environments.


Leann L Norman, Jan Brugués, Kheya Sengupta, Pierre Sens, Helim Aranda-Espinoza
Cell blebbing and membrane area homeostasis in spreading and retracting cells.
Biophys J, 99(6) 1726-1733 (2010)
DOI
Cells remodel their plasma membrane and cytoskeleton during numerous physiological processes, including spreading and motility. Morphological changes require the cell to adjust its membrane tension on different timescales. While it is known that endo- and exocytosis regulate the cell membrane area in a timescale of 1 h, faster processes, such as abrupt cell detachment, require faster regulation of the plasma membrane tension. In this article, we demonstrate that cell blebbing plays a critical role in the global mechanical homeostasis of the cell through regulation of membrane tension. Abrupt cell detachment leads to pronounced blebbing (which slow detachment does not), and blebbing decreases with time in a dynamin-dependent fashion. Cells only start spreading after a lag period whose duration depends on the cell's blebbing activity. Our model quantitatively reproduces the monotonic decay of the blebbing activity and accounts for the lag phase in the spreading of blebbing cells.


Jan Brugués, Benoît Maugis, Jaume Casademunt, Pierre Nassoy, François Amblard, Pierre Sens
Dynamical organization of the cytoskeletal cortex probed by micropipette aspiration.
Proc Natl Acad Sci U.S.A., 107(35) 15415-15420 (2010)
DOI
Bleb-based cell motility proceeds by the successive inflation and retraction of large spherical membrane protrusions ("blebs") coupled with substrate adhesion. In addition to their role in motility, cellular blebs constitute a remarkable illustration of the dynamical interactions between the cytoskeletal cortex and the plasma membrane. Here we study the bleb-based motions of Entamoeba histolytica in the constrained geometry of a micropipette. We construct a generic theoretical model that combines the polymerization of an actin cortex underneath the plasma membrane with the myosin-generated contractile stress in the cortex and the stress-induced failure of membrane-cortex adhesion. One major parameter dictating the cell response to micropipette suction is the stationary cortex thickness, controlled by actin polymerization and depolymerization. The other relevant physical parameters can be combined into two characteristic cortex thicknesses for which the myosin stress (i) balances the suction pressure and (ii) provokes membrane-cortex unbinding. We propose a general phase diagram for cell motions inside a micropipette by comparing these three thicknesses. In particular, we theoretically predict and experimentally verify the existence of saltatory and oscillatory motions for a well-defined range of micropipette suction pressures.


Jan Brugués, Daniel Needleman
Nonequilibrium fluctuations in metaphase spindles: polarized light microscopy, image registration, and correlation functions
Proc SPIE, 7618 Art. No. 76180L (2010)


2009
Jan Brugués, Jaume Casademunt
Self-organization and cooperativity of weakly coupled molecular motors under unequal loading.
Phys Rev Lett, 102(11) 118104-118104 (2009)
We study the collective dynamics of Brownian motors moving on a one-dimensional track when an external load is applied to the leading motor. Motors are driven by a two-state ratchet mechanism, which is appropriate to single-headed kinesins, and their relative motion is only constrained by their mutual interaction potential (weak coupling). We show that unequal loading enhances cooperativity, leading to the formation of clusters with velocities and efficiencies higher than those predicted by simple superposition. When a weak attraction between motors is present, we find nonmonotonic collective velocity-force curves, hysteretic phenomena, and a dynamic self-regulation mechanism that selects the cluster size for optimal performance.


2008
Jan Brugués, Jordi Ignés-Mullol, Jaume Casademunt, Francesc Sagués
Probing elastic anisotropy from defect dynamics in Langmuir monolayers.
Phys Rev Lett, 100(3) 37801-37801 (2008)
We study the dynamics of annihilation of point defects in Langmuir monolayers. The absence of hydrodynamic effects allows us to quantitatively relate the asymmetry in defect mobility to the elastic anisotropy of the material, which in turn can be varied through the control of the surface pressure applied to the monolayer. Using the proposed theoretical analysis, we are able to obtain rather elusive equilibrium properties out of relatively simple dynamical measurements. In particular, we measure the elastic constants and their pressure dependence.


2006
Jan Brugués, Joaquim Gomis, Kiyoshi Kamimura
Newton-Hooke algebras, nonrelativistic branes, and generalized pp-wave metrics
Physical Review D, 73(8) Art. No. 085011 (2006)


2005
Jan Brugués, Joan Rojo, Jorge G. Russo
Nonperturbative states in type II superstring theory from classical spinning membranes
Nucl Phys B, 710(1-2) 117-138 (2005)


2004
Jan Brugués, Thomas Curtright, Joaquim Gomis, Luca Mezincescu
Non-relativistic strings and branes as non-linear realizations of Galilei groups
Phys Lett B, 594(1-2) 227-233 (2004)


2003
Jan Brugués, Joaquim Gomis, T. Mateos, T. Ramirez
Commutative and noncommutative= 2 SYM in (2+ 1) from wrapped D6-branes
Classical And Quantum Gravity, 20 null-null (2003)


2002
Jan Brugués, Joaquim Gomis, T. Mateos, T. Ramirez
Supergravity Duals of Noncommutative Wrapped D6 Branes and Supersymmetry without Supersymmetry
J High Energy Phys, 2002 null-null (2002)