Biology subjects

Derivery, E.

Publications and source records attributed to Derivery, E..

18 recordsLinked to original sources

bioRxiv2026

Asgard archaeal origin of microtubules

Eukaryotic cells change their shapes, actively segregate their DNA and contain membrane networks, facilitated by a complex cytoskeleton containing actin filaments, microtubules made from tubulin, and other components. These filaments have ancient evolutionary origins since actin- and tubulin-like proteins form prokaryotic cytoskeletons in archaea and bacteria. Bona fide eukaryotic F-actin can be traced back to crenarchaea and Asgard archaea, which are the closest known relatives of eukaryotes. A possible Asgard archaeal origin of microtubules was suggested recently with the discovery of a lokiarchaeon containing AtubAB mini microtubules that share architectural features with their eukaryotic counterparts. Using phylogenetic analyses of metagenomic data, here we report the broad occurrence of tubulins in Asgard archaea. Biochemical and structural analyses showed that one of our newly discovered heimdallarchaeial AtubAB tubulin pairs forms four-protofilament mini-microtubules that show dynamic instability and are inhibited by the tubulin drug maytansine. Our work raises the possibility that microtubule architecture and dynamics evolved in Asgard archaea prior to eukaryogenesis.

cell biology↗

bioRxiv2024

Nucleation limited assembly and polarized growth of a de novo-designed allosterically modulatable protein filament

The design of inducibly assembling protein nanomaterials is an outstanding challenge. Here, we describe the computational design of a protein filament formed from a monomeric subunit that binds a peptide ligand. The cryoEM structure of the micron-scale fibers is very close to the computational design model. The ligand acts as a tunable allosteric modulator: while not part of the fiber subunit-subunit interfaces, the assembly of the filament is dependent on ligand addition, with longer peptides having more extensive interaction surfaces with the monomer, promoting more rapid growth. Seeded growth and capping experiments reveal that the filaments grow primarily from one end. We show that designed nucleators that present 12 copies of the peptide ligand promote fiber assembly at concentrations where otherwise assembly occurs very slowly, likely by generating critical local concentrations of monomer in the assembly competent conformation. Following filament assembly, the peptide ligand can be exchanged with free peptide in solution fused to any functional protein of interest, opening the door to a wide variety of tunable engineered materials.

bioengineering↗

bioRxiv2024

Sequence-specific targeting of intrinsically disordered protein regions

Intrinsically disordered proteins and peptides play key roles in biology, but the lack of defined structures and the high variability in sequence and conformational preferences has made targeting such systems challenging. We describe a general approach for designing proteins that bind intrinsically disordered protein regions in diverse extended conformations with side chains fitting into complementary binding pockets. We used the approach to design binders for 39 highly diverse unstructured targets and obtain designs with pM to 100 nM affinities in 34 cases, testing [~]22 designs per target (including polar targets). The designs function in cells and as detection reagents, and are specific for their intended targets in all-by-all binding experiments. Our approach is a major step towards a general solution to the intrinsically disordered protein and peptide recognition problem.

biochemistry↗

bioRxiv2024

Multispectral live-cell imaging with uncompromised spatiotemporal resolution

Multispectral imaging is an established method to extend the number of colours usable in fluorescence imaging beyond the typical limit of three or four, but standard approaches are poorly suited to live-cell imaging. We introduce an approach for multispectral imaging in live cells, comprising an iterative spectral unmixing algorithm and eight channel camera-based image acquisition hardware. This enables the accurate unmixing of low signal-to-noise ratio datasets, typical of live-cell imaging, captured at video rates. We use this approach on a commercial spinning disk confocal microscope and a home-built oblique plane light sheet microscope to image 1-7 spectrally distinct fluorophore species simultaneously, using both fluorescent protein fusions and small-molecule dyes. We further use de novo designed protein-binding proteins (minibinders), labelled with organic fluorophores, and use these in combination with our multispectral imaging approach to study the endosomal trafficking of cell-surface receptors at endogenous levels.

biophysics↗

bioRxiv2023

A force-sensitive mutation reveals a spindle assembly checkpoint-independent role for dynein in anaphase progression

The cytoplasmic dynein-1 (dynein) motor organizes cells by shaping microtubule networks and moving a large variety of cargoes along them. However, dyneins diverse roles complicate in vivo studies of its functions significantly. To address this issue, we have used gene editing to generate a series of missense mutations in Drosophila Dynein heavy chain (Dhc). We find that mutations associated with human neurological disease cause a range of defects in larval and adult flies, including impaired cargo trafficking in neurons. We also describe a novel mutation in the microtubule-binding domain (MTBD) of Dhc that, remarkably, causes metaphase arrest of mitotic spindles in the embryo but does not impair other dynein-dependent processes. We demonstrate that the mitotic arrest is independent of dyneins well-established roles in silencing the spindle assembly checkpoint. In vitro reconstitution and optical trapping assays reveal that the mutation only impairs the performance of dynein under load. In silico all-atom molecular dynamics simulations show that this effect correlates with increased flexibility of the MTBD, as well as an altered orientation of the stalk domain, with respect to the microtubule. Collectively, our data point to a novel role of dynein in anaphase progression that depends on the motor operating in a specific load regime. More broadly, our work illustrates how cytoskeletal transport processes can be dissected in vivo by manipulating mechanical properties of motors.

cell biology↗

bioRxiv2023

Elongator is a microtubule polymerase selective for poly-glutamylated tubulin

Elongator is a tRNA-modifying complex that regulates the fidelity of protein translation. Recently, a moonlighting function of Elongator has been identified in regulating polarization of the microtubule cytoskeleton during asymmetric cell division. Elongator induces symmetry breaking of the anaphase midzone by selectively stabilizing microtubules on one side of the spindle. This polarizes the segregation of signalling endosomes containing cell-fate determinants to only one daughter cell, thus contributing to cell fate determination. Here, we unravelled the molecular mechanism by which Elongator controls microtubule dynamics. Elongator binds simultaneously to the tip of microtubules and also to free GTP-tubulin heterodimers via their C-terminal tails. Elongator thereby locally increases tubulin concentration at microtubule ends, which stabilizes microtubules by increasing their growth speed and decreasing their catastrophe rate. We show that the Elp123 and Elp456 subcomplexes bind to microtubules and free tubulin heterodimers, respectively, and that these activities must be coupled for Elongator to stabilize microtubules. Surprisingly, we found that Elp456 has strong selectivity towards polyglutamylated tubulin dimers. Hence, microtubules assembled by Elongator become selectively enriched with polyglutamylated tubulin. Therefore, Elongator can rewrite the tubulin code of growing microtubules, placing it at the core of cytoskeletal dynamics and polarization during asymmetric cell division.

biochemistry↗

bioRxiv2023

ANXA11 biomolecular condensates facilitate protein-lipid phase coupling on lysosomal membranes

Phase transitions of cellular proteins and lipids play a key role in governing the organisation and coordination of intracellular biology. The frequent juxtaposition of proteinaceous biomolecular condensates to cellular membranes raises the intriguing prospect that phase transitions in proteins and lipids could be co-regulated. Here we investigate this possibility in the ribonucleoprotein (RNP) granule-ANXA11-lysosome ensemble, where ANXA11 tethers RNP granule condensates to lysosomal membranes to enable their co-trafficking. We show that changes to the protein phase state within this system, driven by the low complexity ANXA11 N-terminus, induce a coupled phase state change in the lipids of the underlying membrane. We identify the ANXA11 interacting proteins ALG2 and CALC as potent regulators of ANXA11-based phase coupling and demonstrate their influence on the nanomechanical properties of the ANXA11-lysosome ensemble and its capacity to engage RNP granules. The phenomenon of protein-lipid phase coupling we observe within this system offers an important template to understand the numerous other examples across the cell whereby biomolecular condensates closely juxtapose cell membranes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/533832v1_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@16f3e10org.highwire.dtl.DTLVardef@538f39org.highwire.dtl.DTLVardef@1e00d4eorg.highwire.dtl.DTLVardef@89967d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

HEATR5B associates with dynein-dynactin and selectively promotes motility of AP1-bound endosomal membranes

The dynein motor complex mediates polarised trafficking of a wide variety of organelles, intracellular vesicles and macromolecules. These functions are dependent on the dynactin complex, which helps recruit cargoes to dyneins tail region and activates motor movement. How dynein and dynactin orchestrate trafficking of diverse cargoes is unclear. Here, we identify HEATR5B, an interactor of the AP1 clathrin adaptor complex, as a novel player in dynein-dynactin function. HEATR5B is one of several proteins recovered in a biochemical screen for proteins whose association with the human dynein tail complex is augmented by dynactin. We show that HEATR5B binds directly to the dynein tail and dynactin and stimulates motility of AP1-associated endosomal membranes in human cells. We also demonstrate that the HEATR5B homologue in Drosophila is an essential gene that promotes dynein-based transport of AP1-bound membranes to the Golgi apparatus. As HEATR5B lacks the coiled-coil architecture typical of dynein adaptors, our data point to a non-canonical process orchestrating motor function on a specific cargo. We additionally show that HEATR5B promotes association of AP1 with endosomal membranes in a dynein-independent manner. Thus, HEATR5B co-ordinates multiple events in AP1-based trafficking.

cell biology↗

bioRxiv2023

Structure of the Commander endosomal trafficking complex linked to X-linked intellectual disability/Ritscher-Schinzel syndrome

The Commander complex is required for endosomal recycling of diverse transmembrane cargos and is mutated in Ritscher-Schinzel syndrome. It comprises two subassemblies; Retriever composed of VPS35L, VPS26C and VPS29, and the CCC complex which contains ten subunits COMMD1-COMMD10 and two coiled-coil domain-containing (CCDC) proteins CCDC22 and CCDC93. Combining X-ray crystallography, electron cryomicroscopy and in silico predictions we have assembled a complete structural model of Commander. Retriever is distantly related to the endosomal Retromer complex but has unique features preventing the shared VPS29 subunit from interacting with Retromer-associated factors. The COMMD proteins form a distinctive hetero-decameric ring stabilised by extensive interactions with CCDC22 and CCDC93. These adopt a coiled-coil structure that connects the CCC and Retriever assemblies and recruits a sixteenth subunit, DENND10, to form the complete Commander complex. The structure allows mapping of disease-causing mutations and reveals the molecular features required for the function of this evolutionarily conserved trafficking machinery.

biochemistry↗

bioRxiv2022

De novo design of modular peptide binding proteins by superhelical matching

General approaches for designing sequence-specific peptide binding proteins would have wide utility in proteomics and synthetic biology. Although considerable progress has been made in designing proteins which bind to other proteins, the general peptide binding problem is more challenging as most peptides do not have defined structures in isolation, and to offset the loss in solvation upon binding the protein binding interface has to provide specific hydrogen bonds that complement the majority of the buried peptides backbone polar groups (1-3). Inspired by natural repeat protein-peptide complexes, and engineering efforts to alter their specificity (4-11), we describe a general approach for de novo design of proteins made out of repeating units that bind peptides with repeating sequences such that there is a one to one correspondence between repeat units on the protein and peptide. We develop a rapid docking plus geometric hashing method to identify protein backbones and protein-peptide rigid body arrangements that are compatible with bidentate hydrogen bonds between side chains on the protein and the backbone of the peptide (12); the remainder of the protein sequence is then designed using Rosetta to incorporate additional interactions with the peptide and drive folding to the desired structure. We use this approach to design, from scratch, alpha helical repeat proteins that bind six different tripeptide repeat sequences--PLP, LRP, PEW, IYP, PRM and PKW--in near polyproline 2 helical conformations. The proteins are expressed at high levels in E. coli, are hyperstable, and bind peptides with 4-6 copies of the target tripeptide sequences with nanomolar to picomolar affinities both in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including a ladder of protein sidechain to peptide backbone hydrogen bonds. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating sequences, and for naturally occuring proteins containing disordered regions. Our approach provides a general route to designing specific binding proteins for a broad range of repeating and non-repetitive peptide sequences.

biochemistry↗

bioRxiv2022

Cytomotive actins and tubulins share a polymerisation switch mechanism conferring robust dynamics

Protein filaments are used in myriads of ways to organise other molecules in space and time within cells. Some filament-forming proteins couple the hydrolysis of nucleotides to their polymerisation cycle, thus powering the directed movement of other molecules. These filaments are termed cytomotive. Only members of the actin and tubulin protein superfamilies are known to form cytomotive filaments. We sought to examine the basis of cytomotivity via structural studies of the polymerisation cycles of actin and tubulin homologues from across the tree of life. We analysed published data and performed new structural experiments designed to disentangle functional components of these complex filament systems. In sum, our analysis demonstrates the existence of shared subunit polymerisation switches amongst both cytomotive actins and tubulins, i.e. the conformation of subunits switches upon assembly into filaments. Such cytomotive switches explain filament robustness, by enabling the coupling of kinetic and structural polarities required for useful cytomotive behaviours, and by ensuring that single cytomotive filaments do not fall apart.

molecular biology↗

bioRxiv2022

Induction of cortical Par complex polarity by designed proteins causes cytoskeletal symmetry breaking in unpolarized mammalian cells

Polarized cells rely on a polarized cytoskeleton for polarized trafficking, oriented migration and spindle orientation during asymmetric cell division. While cytoskeleton remodeling machineries have been extensively characterized at the molecular level, how polarity signaling at the cortex controls remodeling of the cytoskeleton in the cytosol remains elusive. In particular, how the Par complex, the conserved mastermind of polarity during asymmetric cell division, gets assembled and functions is not understood at the molecular level. Here, we dissected the logic of the Par complex pathway by capitalizing on designed proteins able to induce spontaneous symmetry breaking of the cortex in populations of naive, unpolarized cells. We found that the primary kinetic barrier to Par complex assembly is the relief of Par6 autoinhibition, and that inducing Par complex cortical polarity was sufficient to induce two key hallmarks of asymmetric cell division in unpolarized cells: spindle orientation and central spindle asymmetry. These two outputs of the Par complex are separately controlled: spindle orientation is determined by Par3 and does not require the kinase activity of aPKC, while central spindle asymmetry solely depends on an asymmetric activity of aPKC at the cortex. Our work shows how polarity information flows between the cortex and the cytosol despite its diffusive nature, and paves the way towards induction of asymmetric cell division in cultured cells.

cell biology↗

bioRxiv2020

Design of Biologically Active Binary Protein 2D Materials

Proteins that assemble into ordered two-dimensional arrays such as S-layers1,2 and designed analogues3-5 have intrigued bioengineers,6,7 but with the exception of a single lattice formed through non-rigid template streptavidin linkers,8 they are constituted from just one protein component. For modulating assembly dynamics and incorporating more complex functionality, materials composed of two components would have considerable advantages.9-12 Here we describe a computational method to generate de-novo binary 2D non-covalent co-assemblies by designing rigid asymmetric interfaces between two distinct protein dihedral building-blocks. The designed array components are soluble at mM concentrations, but when combined at nM concentrations, rapidly assemble into nearly-crystalline micrometer-scale p6m arrays nearly identical to the computational design model in vitro and in cells without the need of a two-dimensional support. Because the material is designed from the ground up, the components can be readily functionalized, and their symmetry reconfigured, enabling formation of ligand arrays with distinguishable surfaces to drive extensive receptor clustering, downstream protein recruitment, and signaling. Using quantitative microscopy we show that arrays assembled on living cells have component stoichiometry and likely structure similar to arrays formed in vitro, suggesting that our material can impose order onto fundamentally disordered substrates like cell membranes. We find further that in sharp contrast to previously characterized cell surface receptor binding assemblies such as antibodies and nanocages, which are rapidly endocytosed, large arrays assembled at the cell surface suppress endocytosis in a tunable manner, with potential therapeutic relevance for extending receptor engagement and immune evasion. Our work paves the way towards synthetic cell biology, where a new generation of multi-protein macroscale materials is designed to modulate cell responses and reshape synthetic and living systems. One Sentence SummaryCo-assembling binary 2D protein crystals enables robust formation of complex large scale ordered biologically active materials

synthetic biology↗

bioRxiv2020

Fibrinogen anchors enable efficient multiplexed patterning of active proteins and subcellular control of membrane-receptor distribution

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, a drawback of current technology is 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.

cell biology↗

bioRxiv2020

The Arp1/11 Minifilament of Dynactin Primes the Endosomal Arp2/3 Complex

Dendritic actin networks develop from a first actin filament through branching by the Arp2/3 complex. At the surface of endosomes, the WASH complex activates the Arp2/3 complex and interacts with the Capping Protein for unclear reasons. Here we show that that the WASH complex interacts with Dynactin and uncaps it through its FAM21 subunit. In vitro, the uncapped Arp1/11 minifilament elongates an actin filament, which then primes the WASH-induced Arp2/3 branching reaction. In Dynactin-depleted cells or in cells where the WASH complex is reconstituted with a FAM21 mutant that cannot uncap Dynactin, formation of branched actin at the endosomal surface is impaired. Our results reveal the importance of the WASH complex in coordinating two complexes containing actin-related proteins. One Sentence SummaryDendritic actin networks grow in an autocatalytic manner starting from the uncapped minifilament of Dynactin.

cell biology↗

bioRxiv2020

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

Between 6-20% of the cellular proteome is under circadian control to tune cell function with cycles of environmental change. For cell viability, and to maintain volume within narrow limits, the osmotic pressure exerted by changes in the soluble proteome must be compensated. The mechanisms and consequences underlying compensation are not known. Here, we show in cultured mammalian cells and in vivo that compensation requires 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 alter their electrical activity at different times of the day. 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.

cell biology↗

bioRxiv2018

Self-repair protects microtubules from their destruction by molecular motors

Microtubules are dynamic polymers that are used for intracellular transport and chromosome segregation during cell division. Their instability stems from the low energy of tubulin dimer interactions, which sets the growing polymer close to its disassembly conditions. Microtubules function in coordination with kinesin and dynein molecular motors, which use ATP hydrolysis to produce mechanical work and move on microtubules. This raises the possibility that the forces produced by walking motors can break dimer interactions and trigger microtubule disassembly. We tested this hypothesis by studying the interplay between microtubules and moving molecular motors in vitro. Our results show that the mechanical work of molecular motors can remove tubulin dimers from the lattice and rapidly destroy microtubules. This effect was not observed when free tubulin dimers were present in the assay. Using fluorescently labelled tubulin dimers we found that dimer removal by motors was compensated for by the insertion of free tubulin dimers into the microtubule lattice. This self-repair mechanism allows microtubules to survive the damage induced by molecular motors as they move along their tracks. Our study reveals the existence of coupling between the motion of kinesin and dynein motors and the renewal of the microtubule lattice.

cell biology↗

bioRxiv2018

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

Three-dimensional neural organoids are emerging tools with the potential for improving our understanding of human brain development and neurological disorders. Recent advances in this field have demonstrated their capacity to model neurogenesis1,2, neuronal migration and positioning3,4, and even response to sensory input5. However, it remains to be seen whether these tissues can model axon guidance dynamics and the formation of complex connectivity with functional neuronal output. Here, we have established a longterm air-liquid interface culture paradigm that leads to improved neuronal survival and allows for imaging of axon guidance. Over time, these cultures spontaneously form thick axon tracts capable of projecting over long distances. Axon bundles display various morphological behaviors including intracortical projection within and across the organoid, growth cone turning, decussation, and projection away from the organoid. Single-cell RNA-sequencing reveals the full repertoire of cortical neuronal identities, and retrograde labelling demonstrates these tract morphologies match the appropriate molecular identities, namely callosal and corticofugal neuron types. We show that these neurons are functionally mature, generate active networks within the organoid, and that extracortical projecting tracts innervate and activate mouse spinal cord-muscle explants. Muscle contractions can be evoked by stimulation of the organoid, while axotomy of the innervating tracts abolishes the muscle contraction response, demonstrating dependence on connection with the organoid. Overall, these results reveal a remarkable selforganization of corticofugal and callosal tracts with a functional output, providing new opportunities to examine relevant aspects of human CNS development and response to injury.

neuroscience↗