TY - JOUR AB - Centrioles are part of centrosomes and cilia, which are microtubule organising centres (MTOC) with diverse functions. Despite their stability, centrioles can disappear during differentiation, such as in oocytes, but little is known about the regulation of their structural integrity. Our previous research revealed that the pericentriolar material (PCM) that surrounds centrioles and its recruiter, Polo kinase, are downregulated in oogenesis and sufficient for maintaining both centrosome structural integrity and MTOC activity. We now show that the expression of specific components of the centriole cartwheel and wall, including ANA1/CEP295, is essential for maintaining centrosome integrity. We find that Polo kinase requires ANA1 to promote centriole stability in cultured cells and eggs. In addition, ANA1 expression prevents the loss of centrioles observed upon PCM-downregulation. However, the centrioles maintained by overexpressing and tethering ANA1 are inactive, unlike the MTOCs observed upon tethering Polo kinase. These findings demonstrate that several centriole components are needed to maintain centrosome structure. Our study also highlights that centrioles are more dynamic than previously believed, with their structural stability relying on the continuous expression of multiple components. AU - Pimenta-Marques, Ana AU - Perestrelo, Tania AU - Dos Reis Rodrigues, Patricia AU - Duarte, Paulo AU - Ferreira-Silva, Ana AU - Lince-Faria, Mariana AU - Bettencourt-Dias, Mónica ID - 14933 IS - 1 JF - EMBO reports TI - Ana1/CEP295 is an essential player in the centrosome maintenance program regulated by Polo kinase and the PCM VL - 25 ER - TY - JOUR AB - Contraction and flow of the actin cell cortex have emerged as a common principle by which cells reorganize their cytoplasm and take shape. However, how these cortical flows interact with adjacent cytoplasmic components, changing their form and localization, and how this affects cytoplasmic organization and cell shape remains unclear. Here we show that in ascidian oocytes, the cooperative activities of cortical actomyosin flows and deformation of the adjacent mitochondria-rich myoplasm drive oocyte cytoplasmic reorganization and shape changes following fertilization. We show that vegetal-directed cortical actomyosin flows, established upon oocyte fertilization, lead to both the accumulation of cortical actin at the vegetal pole of the zygote and compression and local buckling of the adjacent elastic solid-like myoplasm layer due to friction forces generated at their interface. Once cortical flows have ceased, the multiple myoplasm buckles resolve into one larger buckle, which again drives the formation of the contraction pole—a protuberance of the zygote’s vegetal pole where maternal mRNAs accumulate. Thus, our findings reveal a mechanism where cortical actomyosin network flows determine cytoplasmic reorganization and cell shape by deforming adjacent cytoplasmic components through friction forces. AU - Caballero Mancebo, Silvia AU - Shinde, Rushikesh AU - Bolger-Munro, Madison AU - Peruzzo, Matilda AU - Szep, Gregory AU - Steccari, Irene AU - Labrousse Arias, David AU - Zheden, Vanessa AU - Merrin, Jack AU - Callan-Jones, Andrew AU - Voituriez, Raphaël AU - Heisenberg, Carl-Philipp J ID - 14846 JF - Nature Physics SN - 1745-2473 TI - Friction forces determine cytoplasmic reorganization and shape changes of ascidian oocytes upon fertilization ER - TY - JOUR AB - The extracellular matrix (ECM) serves as a scaffold for cells and plays an essential role in regulating numerous cellular processes, including cell migration and proliferation. Due to limitations in specimen preparation for conventional room-temperature electron microscopy, we lack structural knowledge on how ECM components are secreted, remodeled, and interact with surrounding cells. We have developed a 3D-ECM platform compatible with sample thinning by cryo-focused ion beam milling, the lift-out extraction procedure, and cryo-electron tomography. Our workflow implements cell-derived matrices (CDMs) grown on EM grids, resulting in a versatile tool closely mimicking ECM environments. This allows us to visualize ECM for the first time in its hydrated, native context. Our data reveal an intricate network of extracellular fibers, their positioning relative to matrix-secreting cells, and previously unresolved structural entities. Our workflow and results add to the structural atlas of the ECM, providing novel insights into its secretion and assembly. AU - Zens, Bettina AU - Fäßler, Florian AU - Hansen, Jesse AU - Hauschild, Robert AU - Datler, Julia AU - Hodirnau, Victor-Valentin AU - Zheden, Vanessa AU - Alanko, Jonna H AU - Sixt, Michael K AU - Schur, Florian KM ID - 15146 IS - 6 JF - Journal of Cell Biology SN - 0021-9525 TI - Lift-out cryo-FIBSEM and cryo-ET reveal the ultrastructural landscape of extracellular matrix VL - 223 ER - TY - CHAP AB - Imaging of the immunological synapse (IS) between dendritic cells (DCs) and T cells in suspension is hampered by suboptimal alignment of cell-cell contacts along the vertical imaging plane. This requires optical sectioning that often results in unsatisfactory resolution in time and space. Here, we present a workflow where DCs and T cells are confined between a layer of glass and polydimethylsiloxane (PDMS) that orients the cells along one, horizontal imaging plane, allowing for fast en-face-imaging of the DC-T cell IS. AU - Leithner, Alexander F AU - Merrin, Jack AU - Sixt, Michael K ED - Baldari, Cosima ED - Dustin, Michael ID - 13052 SN - 1064-3745 T2 - The Immune Synapse TI - En-Face Imaging of T Cell-Dendritic Cell Immunological Synapses VL - 2654 ER - TY - JOUR AB - The intricate regulatory processes behind actin polymerization play a crucial role in cellular biology, including essential mechanisms such as cell migration or cell division. However, the self-organizing principles governing actin polymerization are still poorly understood. In this perspective article, we compare the Belousov-Zhabotinsky (BZ) reaction, a classic and well understood chemical oscillator known for its self-organizing spatiotemporal dynamics, with the excitable dynamics of polymerizing actin. While the BZ reaction originates from the domain of inorganic chemistry, it shares remarkable similarities with actin polymerization, including the characteristic propagating waves, which are influenced by geometry and external fields, and the emergent collective behavior. Starting with a general description of emerging patterns, we elaborate on single droplets or cell-level dynamics, the influence of geometric confinements and conclude with collective interactions. Comparing these two systems sheds light on the universal nature of self-organization principles in both living and inanimate systems. AU - Riedl, Michael AU - Sixt, Michael K ID - 14555 JF - Frontiers in Cell and Developmental Biology TI - The excitable nature of polymerizing actin and the Belousov-Zhabotinsky reaction VL - 11 ER - TY - THES AB - Most motions of many-body systems at any scale in nature with sufficient degrees of freedom tend to be chaotic; reaching from the orbital motion of planets, the air currents in our atmosphere, down to the water flowing through our pipelines or the movement of a population of bacteria. To the observer it is therefore intriguing when a moving collective exhibits order. Collective motion of flocks of birds, schools of fish or swarms of self-propelled particles or robots have been studied extensively over the past decades but the mechanisms involved in the transition from chaos to order remain unclear. Here, the interactions, that in most systems give rise to chaos, sustain order. In this thesis we investigate mechanisms that preserve, destabilize or lead to the ordered state. We show that endothelial cells migrating in circular confinements transition to a collective rotating state and concomitantly synchronize the frequencies of nucleating actin waves within individual cells. Consequently, the frequency dependent cell migration speed uniformizes across the population. Complementary to the WAVE dependent nucleation of traveling actin waves, we show that in leukocytes the actin polymerization depending on WASp generates pushing forces locally at stationary patches. Next, in pipe flows, we study methods to disrupt the self--sustaining cycle of turbulence and therefore relaminarize the flow. While we find in pulsating flow conditions that turbulence emerges through a helical instability during the decelerating phase. Finally, we show quantitatively in brain slices of mice that wild-type control neurons can compensate the migratory deficits of a genetically modified neuronal sub--population in the developing cortex. AU - Riedl, Michael ID - 14530 KW - Synchronization KW - Collective Movement KW - Active Matter KW - Cell Migration KW - Active Colloids SN - 2663 - 337X TI - Synchronization in collectively moving active matter ER - TY - JOUR AB - Whether one considers swarming insects, flocking birds, or bacterial colonies, collective motion arises from the coordination of individuals and entails the adjustment of their respective velocities. In particular, in close confinements, such as those encountered by dense cell populations during development or regeneration, collective migration can only arise coordinately. Yet, how individuals unify their velocities is often not understood. Focusing on a finite number of cells in circular confinements, we identify waves of polymerizing actin that function as a pacemaker governing the speed of individual cells. We show that the onset of collective motion coincides with the synchronization of the wave nucleation frequencies across the population. Employing a simpler and more readily accessible mechanical model system of active spheres, we identify the synchronization of the individuals’ internal oscillators as one of the essential requirements to reach the corresponding collective state. The mechanical ‘toy’ experiment illustrates that the global synchronous state is achieved by nearest neighbor coupling. We suggest by analogy that local coupling and the synchronization of actin waves are essential for the emergent, self-organized motion of cell collectives. AU - Riedl, Michael AU - Mayer, Isabelle D AU - Merrin, Jack AU - Sixt, Michael K AU - Hof, Björn ID - 14361 JF - Nature Communications TI - Synchronization in collectively moving inanimate and living active matter VL - 14 ER - TY - JOUR AB - To navigate through diverse tissues, migrating cells must balance persistent self-propelled motion with adaptive behaviors to circumvent obstacles. We identify a curvature-sensing mechanism underlying obstacle evasion in immune-like cells. Specifically, we propose that actin polymerization at the advancing edge of migrating cells is inhibited by the curvature-sensitive BAR domain protein Snx33 in regions with inward plasma membrane curvature. The genetic perturbation of this machinery reduces the cells’ capacity to evade obstructions combined with faster and more persistent cell migration in obstacle-free environments. Our results show how cells can read out their surface topography and utilize actin and plasma membrane biophysics to interpret their environment, allowing them to adaptively decide if they should move ahead or turn away. On the basis of our findings, we propose that the natural diversity of BAR domain proteins may allow cells to tune their curvature sensing machinery to match the shape characteristics in their environment. AU - Sitarska, Ewa AU - Almeida, Silvia Dias AU - Beckwith, Marianne Sandvold AU - Stopp, Julian A AU - Czuchnowski, Jakub AU - Siggel, Marc AU - Roessner, Rita AU - Tschanz, Aline AU - Ejsing, Christer AU - Schwab, Yannick AU - Kosinski, Jan AU - Sixt, Michael K AU - Kreshuk, Anna AU - Erzberger, Anna AU - Diz-Muñoz, Alba ID - 14360 JF - Nature Communications TI - Sensing their plasma membrane curvature allows migrating cells to circumvent obstacles VL - 14 ER - TY - JOUR AB - Immune responses rely on the rapid and coordinated migration of leukocytes. Whereas it is well established that single-cell migration is often guided by gradients of chemokines and other chemoattractants, it remains poorly understood how these gradients are generated, maintained, and modulated. By combining experimental data with theory on leukocyte chemotaxis guided by the G protein–coupled receptor (GPCR) CCR7, we demonstrate that in addition to its role as the sensory receptor that steers migration, CCR7 also acts as a generator and a modulator of chemotactic gradients. Upon exposure to the CCR7 ligand CCL19, dendritic cells (DCs) effectively internalize the receptor and ligand as part of the canonical GPCR desensitization response. We show that CCR7 internalization also acts as an effective sink for the chemoattractant, dynamically shaping the spatiotemporal distribution of the chemokine. This mechanism drives complex collective migration patterns, enabling DCs to create or sharpen chemotactic gradients. We further show that these self-generated gradients can sustain the long-range guidance of DCs, adapt collective migration patterns to the size and geometry of the environment, and provide a guidance cue for other comigrating cells. Such a dual role of CCR7 as a GPCR that both senses and consumes its ligand can thus provide a novel mode of cellular self-organization. AU - Alanko, Jonna H AU - Ucar, Mehmet C AU - Canigova, Nikola AU - Stopp, Julian A AU - Schwarz, Jan AU - Merrin, Jack AU - Hannezo, Edouard B AU - Sixt, Michael K ID - 14274 IS - 87 JF - Science Immunology KW - General Medicine KW - Immunology SN - 2470-9468 TI - CCR7 acts as both a sensor and a sink for CCL19 to coordinate collective leukocyte migration VL - 8 ER - TY - THES AU - Stopp, Julian A ID - 14697 SN - 2663 - 337X TI - Neutrophils on the hunt: Migratory strategies employed by neutrophils to fulfill their effector function ER -