High-efficacy subcellular micropatterning of proteins using fibrinogen anchors.

Protein micropatterning allows proteins to be precisely deposited onto a substrate of choice and is now routinely used in cell biology and in vitro reconstitution. However, drawbacks of current technology are that micropatterning efficiency can be variable between proteins and that proteins may lose activity on the micropatterns. Here, we describe a general method to enable micropatterning of virtually any protein at high specificity and homogeneity while maintaining its activity. Our method is based on an anchor that micropatterns well, fibrinogen, which we functionalized to bind to common purification tags. This enhances micropatterning on various substrates, facilitates multiplexed micropatterning, and dramatically improves the on-pattern activity of fragile proteins like molecular motors. Furthermore, it enhances the micropatterning of hard-to-micropattern cells. Last, this method enables subcellular micropatterning, whereby complex micropatterns simultaneously control cell shape and the distribution of transmembrane receptors within that cell. Altogether, these results open new avenues for cell biology

Cerebral organoids at the air-liquid interface generate diverse nerve tracts with functional output.

Neural organoids have the potential to improve our understanding of human brain development and neurological disorders. However, it remains to be seen whether these tissues can model circuit formation with functional neuronal output. Here we have adapted air-liquid interface culture to cerebral organoids, leading to improved neuronal survival and axon outgrowth. The resulting thick axon tracts display various morphologies, including long-range projection within and away from the organoid, growth-cone turning, and decussation. Single-cell RNA sequencing reveals various cortical neuronal identities, and retrograde tracing demonstrates tract morphologies that match proper molecular identities. These cultures exhibit active neuronal networks, and subcortical projecting tracts can innervate mouse spinal cord explants and evoke contractions of adjacent muscle in a manner dependent on intact organoid-derived innervating tracts. Overall, these results reveal a remarkable self-organization of corticofugal and callosal tracts with a functional output, providing new opportunities to examine relevant aspects of human CNS development and disease

Actin Polymerization Controls the Organization of WASH Domains at the Surface of Endosomes

Sorting of cargoes in endosomes occurs through their selective enrichment into sorting platforms, where transport intermediates are generated. The WASH complex, which directly binds to lipids, activates the Arp2/3 complex and hence actin polymerization onto such sorting platforms. Here, we analyzed the role of actin polymerization in the physiology of endosomal domains containing WASH using quantitative image analysis. Actin depolymerization is known to enlarge endosomes. Using a novel colocalization method that is insensitive to the heterogeneity of size and shape of endosomes, we further show that preventing the generation of branched actin networks induces endosomal accumulation of the WASH complex. Moreover, we found that actin depolymerization induces a dramatic decrease in the recovery of endosomal WASH after photobleaching. This result suggests a built-in turnover, where the actin network, i.e. the product of the WASH complex, contributes to the dynamic exchange of the WASH complex by promoting its detachment from endosomes. Our experiments also provide evidence for a role of actin polymerization in the lateral compartmentalization of endosomes: several WASH domains exist at the surface of enlarged endosomes, however, the WASH domains coalesce upon actin depolymerization or Arp2/3 depletion. Branched actin networks are thus involved in the regulation of the size of WASH domains. The potential role of this regulation in membrane scission are discussed

Free Brick1 Is a Trimeric Precursor in the Assembly of a Functional Wave Complex

Background: The Wave complex activates the Arp2/3 complex, inducing actin polymerization in lamellipodia and membrane ruffles. The Wave complex is composed of five subunits, the smallest of which, Brick1/Hspc300 (Brk1), is the least characterized. We previously reported that, unlike the other subunits, Brk1 also exists as a free form. Principal Findings: Here we report that this free form of Brk1 is composed of homotrimers. Using a novel assay in which purified free Brk1 is electroporated into HeLa cells, we were able to follow its biochemical fate in cells and to show that free Brk1 becomes incorporated into the Wave complex. Importantly, incorporation of free Brk1 into the Wave complex was blocked upon inhibition of protein synthesis and incorporated Brk1 was found to associate preferentially with neosynthesized subunits. Brk1 depleted HeLa cells were found to bleb, as were Nap1, Wave2 or ARPC2 depleted cells, suggesting that this blebbing phenotype of Brk1 depleted cells is due to an impairment of the Wave complex function rather than a specific function of free Brk1. Blebs of Brk1 depleted cells were emitted at sites where lamellipodia and membrane ruffles were normally emitted. In Brk1 depleted cells, the electroporation of free Brk1 was sufficient to restore Wave complex assembly and to rescue the blebbing phenotype. Conclusion: Together these results establish that the free form of Brk1 is an essential precursor in the assembly of

Compensatory ion transport buffers daily protein rhythms to regulate osmotic balance and cellular physiology

Abstract: Between 6–20% of the cellular proteome is under circadian control and tunes mammalian cell function with daily environmental cycles. For cell viability, and to maintain volume within narrow limits, the daily variation in osmotic potential exerted by changes in the soluble proteome must be counterbalanced. The mechanisms and consequences of this osmotic compensation have not been investigated before. In cultured cells and in tissue we find that compensation involves electroneutral active transport of Na+, K+, and Cl− through differential activity of SLC12A family cotransporters. In cardiomyocytes ex vivo and in vivo, compensatory ion fluxes confer daily variation in electrical activity. Perturbation of soluble protein abundance has commensurate effects on ion composition and cellular function across the circadian cycle. Thus, circadian regulation of the proteome impacts ion homeostasis with substantial consequences for the physiology of electrically active cells such as cardiomyocytes

Remodeling of biological membranes by molecular machines activating the Arp2/3 complex

La polymérisation de filaments d'actine a souvent lieu à la surface de membranes, générant des forces capables de les remodeler. Le complexe Arp2/3 induit la formation de réseaux d'actine branchée lorsqu'il est activé par des protéines NPF (Nucleation promoting factor). Dans cette thèse, nous nous sommes intéressés aux protéines NPF WAVE et WASH. Les protéines WAVE sont requises lors de la formation des lamellipodes et font partie d'un complexe multiprotéique comportant 5 sous-unités. Nous avons développé une nouvelle méthode de purification du complexe WAVE grâce à laquelle nous avons montré que ce complexe est intrinsèquement inactif. Ceci permet de mieux comprendre comment l'activité WAVE est régulée au niveau moléculaire. Brk1 est la seule sous-unité du complexe WAVE à exister sous une forme libre, et nous avons montré que cette forme de Brk1 est un précurseur lors de l'assemblage du complexe WAVE. Ceci indique que l'assemblage du complexe WAVE est un processus finement contrôlé. Dans un deuxième temps, nous nous sommes intéressés au rôle cellulaire de WASH, jusqu alors inconnu. Nous avons montré que WASH est spécifiquement localisé au niveau de domaines membranaires des endosomes de tri, et que la polymérisation d'actine induite par WASH est requise pour l'étape de scission lors de la formation d'intermédiaires de transport. De plus, nous avons montré que la dépolymérisation de l actine induit la fusion des domaines WASH à la surface des endosomes, ce qui indique un rôle actif du cytosquelette d actine dans la compartimentalisation de la membrane des endosomes. L ensemble de ces résultats révèle ainsi l importance du cytosquelette d actine dans le tri endosomal.The polarized assembly of actin filaments often occurs at the surface of membranes, developing forces that cause their deformation or movement. The Arp2/3 complex generates branched actin networks when activated by Nucleation Promoting Factors (NPF). This work focuses on two NPFs: WAVE and WASH. WAVE proteins are required for lamellipodia formation and are embedded into a 5-subunit multiprotein complex. An actual challenge is to understand how this complex regulates their activity. Using a novel purification method that we developed, we showed that the WAVE complex is intrinsically inactive, thus providing molecular basis to understand how WAVE activity is regulated during cell migration. The Brk1 subunit is the only subunit of the WAVE complex to also exist in a free pool in cells. We discovered that this free pool is a precursor in the assembly of the WAVE complex, highlighting the need of a specific assembly pathway for this complex. Then, we characterized the cellular role of the NPF WASH, which was previously elusive. We showed that WASH localizes to domains of endosome membranes and that actin polymerization by the WASH pathway is required for efficient fission of transport intermediates from endosomes. Furthermore, we showed that actin polymerization prevents the fusion of WASH domains at the surface of endosomes, suggesting an active role of the actin cytoskeleton in the compartmentalization of endosome membranes. All together, these results shed light on the elusive role of endosomal actin.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF