Biology subjects

Kim, H.

Publications and source records attributed to Kim, H..

At least 343 records · Page 19Linked to original sources

bioRxiv2022

Foldcomp: a library and format for compressing and indexing large protein structure sets

Highly accurate protein structure predictors have generated hundreds of millions of protein structures; these pose a challenge in terms of storage and processing. Here we present Foldcomp, a novel lossy structure compression algorithm and indexing system to address this challenge. By using a combination of internal and cartesian coordinates and a bi-directional NeRF-based strategy, Foldcomp improves the compression ratio by a factor of 3 compared to the next best method. Its reconstruction error of 0.08[A] is comparable to the best lossy compressor. It is 5 times faster than the next fastest compressor and competes with the fastest decompressors. With its multi-threading implementation and a Python interface that allows for easy database downloads and efficient querying of protein structures by accession, Foldcomp is a powerful tool for managing and analyzing large collections of protein structures. AvailabilityFoldcomp is a free open-source library and command-line software available for Linux, macOS and Windows at https://foldcomp.foldseek.com. Foldcomp provides the AlphaFold Swiss-Prot (2.9GB), TrEMBL (1.1TB) and ESMatlas HQ (114GB) database ready-for-download.

bioinformatics↗

bioRxiv2022

Integrated proteomics reveals the landscape of autophagy degradation in human neurons and autophagy receptors regulating neuronal activity

Autophagy is a catabolic and self-degradative process crucial for maintaining cellular homeostasis. Malfunctional autophagy is implicated in neurodevelopmental and neurodegenerative diseases. However, the exact role and targets of autophagy in human neurons remain elusive. Here we reported a systematic investigation of neuronal autophagy targets through integrated proteomics. Deep proteomic profiling of multiple autophagy-deficient lines of human induced neurons, mouse brains, and LC3-interactome uncovers a role of neuronal autophagy in targeting primarily endoplasmic reticulum (ER), mitochondria, endosome, Golgi apparatus, synaptic vesicle (SV) proteins, and cAMP-PKA pathway, for degradation. Tubular ER and specific SV proteins are significant autophagy cargos in the axons. Functional validation identified calumenin as an ER resident autophagy receptor and illuminated a role of autophagy in regulating PKA and neuronal activity through AKAP11-mediated degradation. Our study thus reveals the landscape of autophagy degradation in human neurons and offers molecular insight into mechanisms of neurological disorders linked to autophagy deficiency. HighlightO_LIIntegrated proteomics reveals the landscape of autophagy degradation in human neurons C_LIO_LIAutophagy clears tubular ER and selective ER and synaptic vesicle proteins in neurons C_LIO_LICalumenin is an ER resident autophagy receptor C_LIO_LIAutophagy controls PKA pathway and neuronal activity through autophagy receptor AKAP11 C_LI

neuroscience↗

bioRxiv2022

Single-Cell Transcriptional and Epigenetic Profiles of Male Breast Cancer Nominate Salient Cancer-Specific Enhancers

Male breast cancer represents about 1% of all breast cancer diagnoses and, although there are some similarities between male and female breast cancer, the paucity of data available on male breast cancer makes it difficult to establish targeted therapies. To date, most male breast cancers (MBC) are treated according to protocols established for female breast cancer (FBC). Thus, defining the transcriptional and epigenetic landscape of MBC with improved resolution is critical for developing better avenues for therapeutic intervention. In this study, we present matched transcriptional (scRNA-seq) and epigenetic (scATAC-seq) profiles at single-cell resolution of two treatment naive MBC tumors processed immediately after surgical resection. These data enable the detection of differentially expressed genes between male and female breast tumors across immune, stromal, and malignant cell types, to highlight several genes that may have therapeutic implications. Notably, MYC target genes and mTORC1 signaling genes were significantly upregulated in the malignant cells of MBC compared to the female counterparts. To understand how the regulatory landscape of MBC give rise to these male-specific gene expression patterns, we leveraged the scATAC-seq data to systematically link changes in chromatin accessibility to changes in gene expression within each cell type. We observed cancer-specific rewiring of several salient enhancers and posit that these enhancers have a higher regulatory load than lineage specific enhancers. We highlight two examples of previously unannotated cancer-cell specific enhancers of ANXA2 and PRDX4 gene expression and show evidence for super-enhancer regulation of LAMB3 and CD47 in male breast cancer cells. Overall, this dataset annotates clinically relevant regulatory networks in male breast tumors, providing a useful resource that expands our current understanding of the gene expression programs that underlie the biology of MBC.

genomics↗

bioRxiv2022

Hippocampal engram networks for fear memory recruit new synapses and modify pre-existing synapses in vivo.

As basic units of neural networks, ensembles of synapses underlie cognitive functions like learning and memory. These synaptic engrams show elevated synaptic density among engram cells following contextual fear memory formation. Subsequent analysis of the CA3-CA1 engram synapse revealed larger spine sizes, as the synaptic connectivity correlated to the memory strength. Here, we elucidate the synapse dynamics between CA3 and CA1 by tracking identical synapses at multiple time points by adapting two-photon microscopy and dual-eGRASP technique in vivo. After memory formation, synaptic connections between engram populations are enhanced in conjunction with synaptogenesis within the hippocampal network. However, extinction learning specifically correlated with the disappearance of CA3 engram to CA1 engram (E-E) synapses. We observed "newly formed" synapses near pre-existing synapses, which clustered CA3-CA1 engram synapses after fear memory formation. Overall, we conclude that dynamics at CA3 to CA1 E-E synapses are key sites for modification during fear memory states. HighlightsO_LIWe adapted four-color two-photon microscopy with dual-eGRASP for a longitudinal observation of synaptic connections between CA3 and CA1. C_LIO_LISynaptogenesis according to fear memory formation was specifically observed in E-E synapses. C_LIO_LIExtinction learning significantly correlated with the disappearance of E-E synapses. C_LIO_LIParticular distribution pattern of newly formed E-E synapses was observed after memory formation. C_LI

neuroscience↗

bioRxiv2022

IMPASTO: Multiplexed cyclic imaging without signal removal via self-supervised neural unmixing

Spatially resolved proteomics requires a highly multiplexed imaging modality. Cyclic imaging techniques, which repeat staining, imaging, and signal erasure, have been adopted for this purpose. However, due to tissue distortion, it is challenging to obtain high fluorescent signal intensities and complete signal erasure in thick tissue with cyclic imaging techniques. Here, we propose an "erasureless" cyclic imaging method named IMPASTO. In IMPASTO, specimens are iteratively stained and imaged without signal erasure. Then, images from two consecutive rounds are unmixed to retrieve the images of single proteins through self-supervised machine learning without any prior training. Using IMPASTO, we demonstrate 30-plex imaging from brain slices in 10 rounds, and when used in combination with spectral unmixing, in five rounds. We show that IMPASTO causes negligible tissue distortion and demonstrate 3D multiplexed imaging of brain slices. Further, we show that IMPASTO can shorten the signal removal processes of existing cyclic imaging techniques.

bioengineering↗

bioRxiv2022

Esr1+ hypothalamic-habenula neurons shape aversive states.

Excitatory projections from the lateral hypothalamic area (LHA) to the lateral habenula (LHb) drive aversive responses. We used Patch-seq guided multimodal classification to define the structural and functional heterogeneity of the LHA-LHb pathway. Our classification identified six glutamatergic neuron types with unique electrophysiological properties, molecular profiles, and projection patterns. We found that genetically-defined LHA-LHb neurons signal distinct aspects of emotional or naturalistic behaviors: Esr1+ LHA-LHb neurons induce aversion, whereas Npy+ LHA-LHb neurons control rearing behavior. Repeated optogenetic drive of Esr1+ LHA-LHb neurons induces a behaviorally persistent aversive state, and large-scale recordings showed a region-specific neural representation of the aversive state in the prelimbic region of the prefrontal cortex. We further found that exposure to unpredictable mild shocks induced a sex-specific sensitivity to develop a stress state in female mice, which was associated with a specific shift in the intrinsic properties of bursting-type Esr1+ LHA-LHb neurons. In summary, we describe the diversity of LHA-LHb neuron types, and provide evidence for the role of Esr1+ neurons in aversion and sexually dimorphic stress sensitivity.

neuroscience↗

bioRxiv2022

A Model of High-Speed Endovascular Sonothrombolysis with Vortex Ultrasound-Induced Shear Stress to Treat Cerebral Venous Sinus Thrombosis

This research aims to demonstrate a novel vortex ultrasound enabled endovascular thrombolysis method designed for treating cerebral venous sinus thrombosis (CVST). This is a topic of significant importance since current treatment modalities for CVST still fail in as many as 20-40% of the cases and the incidence of CVST has increased since the outbreak of the COVID-19 pandemic. Compared with conventional anticoagulant or thrombolytic drugs, sonothrombolysis has the potential to remarkably shorten the required treatment time owing to the direct clot targeting with acoustic waves. However, previously reported strategies for sonothrombolysis have not demonstrated clinically meaningful outcomes (e.g., recanalization within 30 minutes) in treating large, completely occluded veins or arteries. In this paper, we demonstrated a new vortex ultrasound technique for endovascular sonothrombolysis utilizing wave-matter interaction-induced shear stress to enhance the lytic rate substantially. Our in vitro experiment showed that the lytic rate was increased by at least 64.3 % compared with the nonvortex endovascular ultrasound treatment. A 3.1 g, 7.5 cm long, completely occluded in vitro 3D model of acute CVST was fully recanalized within 8 minutes with a record-high lytic rate of 237.5 mg/min for acute bovine clot in vitro. Furthermore, we confirmed that the vortex ultrasound causes no vessel wall damage over ex vivo bovine veins. This vortex ultrasound thrombolysis technique potentially presents a new life-saving tool for severe CVST cases that cannot be efficaciously treated using existing therapies.

bioengineering↗

bioRxiv2022

Prevention of thrombocytopenia and thrombosis in heparin-induced thrombocytopenia (HIT) using deglycosylated KKO: A novel therapeutic?

Heparin-induced thrombocytopenia (HIT) is characterized by mild thrombocytopenia associated with a highly prothrombotic state due to the development of pathogenic antibodies that recognize human (h) platelet factor 4 (PF4) complexed with various polyanions. While non-heparin anticoagulants and intravenous immunoglobulin (IVIG) are the mainstay of care, bleeding may develop, and risk of new thromboembolic events remain. We had described a mouse IgG{kappa}2b antibody KKO that mimics the sentinel features of pathogenic HIT antibodies, including binding to the same neoepitope on hPF4:polyanion complexes. KKO, like HIT IgGs, activates platelets through Fc{gamma}RIIA and induces complement activation. We now asked whether Fc-modified KKO can be used as a novel therapeutic to prevent or treat HIT. Using the endoglycosidase EndoS, we created deglycosylated KKO (DGKKO). DGKKO bound to PF4-polyanion complexes, and blocked Fc{gamma}RIIA-dependent activation of PF4 treated platelets by KKO, 5B9 (another HIT-like monoclonal antibody), and isolated IgGs from HIT patients. DGKKO also decreased complement activation and deposition of C3c on platelets. Injection of DGKKO into "HIT mice" lacking mouse PF4, but transgenic for hPF4 and Fc{gamma}RIIA, prevented and reversed thrombocytopenia when injected before or after KKO, 5B9 or HIT IgG, respectively, in a microfluidic system. DGKKO reversed antibody-induced thrombus growth in HIT mice. In contrast, DGKKO was ineffective in preventing thrombosis by IgG from a patient with the HIT-related disorder, vaccine-induced immune thrombotic thrombocytopenia. Thus, DGKKO may represent a new class of therapeutics for targeted treatment of patients with HIT. Key PointsO_LIDeglycosylated (DG) KKO can reverse thrombocytopenia in a HIT murine model. C_LIO_LIDGKKO can prevent/reverse thrombosis in vitro and in a HIT murine model. C_LI

cell biology↗

bioRxiv2022

Generalized strategy for engineering mammalian cell-compatible RNA-based biosensors from random sequence libraries

Fluorescent RNA-based biosensors are useful tools for real-time detection of molecules in living cells. These biosensors typically consist of a chromophore-binding aptamer and a target-binding aptamer, whereby the chromophore-binding aptamer is destabilized until a target is captured, which causes a conformational change to permit chromophore binding and an increase in fluorescence. The target-binding region is typically fabricated using known riboswitch motifs, which are already known to have target specificity and undergo structural changes upon binding. However, known riboswitches only exist for a limited number of molecules, significantly constraining biosensor design. To overcome this challenge, we designed a framework for producing mammalian cell-compatible biosensors using aptamers selected from a large random library by capture-SELEX. As a proof-of-concept, we generated and characterized a fluorescent RNA biosensor against L-dopa, the precursor of several neurotransmitters. Overall, we suggest that this approach will have utility for generating RNA biosensors that can reliably detect custom targets in mammalian cells.

molecular biology↗

bioRxiv2022

Depletion Assisted Hemin Affinity (DAsHA) Proteomics Reveals an Expanded Landscape of Heme Binding Proteins

Heme b (iron protoporphyrin IX) plays important roles in biology as a metallocofactor and signaling molecule. However, the targets of heme signaling and the network of proteins that mediate the exchange of heme from sites of synthesis or uptake to heme dependent or regulated proteins are poorly understood. Herein, we describe a quantitative mass spectrometry-based chemoproteomics strategy to identify exchange labile hemoproteins in human embryonic kidney HEK293 cells that may be relevant to heme signaling and trafficking. The strategy involves depleting endogenous heme with the heme biosynthetic inhibitor succinylacetone (SA), leaving putative heme binding proteins in their apo-state, followed by the capture of those proteins using hemin-agarose resin and finally elution and identification by mass spectrometry. By identifying only those proteins that interact with high specificity to hemin-agarose relative to control beaded agarose in a SA-dependent manner, we have expanded the number of proteins and ontologies that may be involved in binding and buffering labile heme or are targets of heme signaling. Notably, these include proteins involved in chromatin remodeling, DNA damage response, RNA splicing, cytoskeletal organization and vesicular trafficking, many of which have been associated with heme through complimentary studies published recently. Taken together, these results provide support for the emerging role for heme in an expanded set of cellular processes from genome integrity to protein trafficking and beyond.

biochemistry↗

bioRxiv2022

Live Cell Lineage Tracing of Dormant Cancer Cells

Breast cancer is a leading cause of global cancer-related deaths, and metastasis is the overwhelming culprit of poor patient prognosis. The most nefarious aspect of metastasis is dormancy, a prolonged period between primary tumor resection and relapse. Current therapies are insufficient at killing dormant cells; thus, they can remain quiescent in the body for decades until eventually undergoing a phenotypic switch, resulting in metastases that are more adaptable and more drug resistant. Unfortunately, dormancy has few in vitro models, largely because lab-derived cell lines are highly proliferative. Existing models address tumor dormancy, not cellular dormancy, because tracking individual cells is technically challenging. To combat this problem, we adapted a live cell lineage approach to find and track individual dormant cells, distinguishing them from proliferative and dying cells over multiple days. We applied this approach across a range of different in vitro microenvironments. Our approach revealed that the proportion of cells that exhibited long-term quiescence was regulated by both cell intrinsic and extrinsic factors, with the most dormant cells found in 3D collagen gels. We hope this approach will prove useful to biologists and bioengineers in the dormancy community to identify, quantify, and study dormant tumor cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/511405v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@5d0af9org.highwire.dtl.DTLVardef@7b7830org.highwire.dtl.DTLVardef@f9fc4org.highwire.dtl.DTLVardef@1c20a39_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Synapsing with oligodendrocyte precursor cells stop sensory axons regenerating into the spinal cord

Primary sensory axons stop regenerating as they re-enter the spinal cord, resulting in incurable sensory loss. What arrests them has remained unclear. We previously showed that axons stop by forming synaptic contacts with unknown non-neuronal cells. Here, we identified these cells in adult mice as oligodendrocyte precursor cells (OPCs). We also found that only a few axons stop regenerating by forming dystrophic endings, exclusively at the CNS:PNS borderline where OPCs are absent. Most axons stop in contacts with a dense network of OPC processes. Live imaging, immuno-EM and OPC-DRG co-culture additionally suggest that axons are rapidly immobilized by forming synapses with OPCs. Genetic OPC ablation enables many axons to continue regenerating deep into the spinal cord. We propose that sensory axons stop regenerating by encountering OPCs that induce presynaptic differentiation. Our findings identify OPCs as a major regenerative barrier that prevents intraspinal restoration of sensory circuits following spinal root injury.

neuroscience↗

bioRxiv2022

SARS-CoV-2 variants show temperature-dependent enhancedpolymerase activity in the upper respiratory tract and high transmissibility

With the convergent global emergence of SARS-CoV-2 variants of concern (VOC), a precise comparison study of viral fitness and transmission characteristics is necessary for the prediction of dominant VOCs and the development of suitable countermeasures. While airway temperature plays important roles in the fitness and transmissibility of respiratory tract viruses, it has not been well studied with SARS-CoV-2. Here we demonstrate that natural temperature differences between the upper (33{degrees}C) and lower (37{degrees}C) respiratory tract have profound effects on SARS-CoV-2 replication and transmission. Specifically, SARS-COV-2 variants containing the P323L or P323L/G671S mutation in the NSP12 RNA-dependent RNA polymerase (RdRp) exhibited enhanced RdRp enzymatic activity at 33{degrees}C compared to 37{degrees}C and high transmissibility in ferrets. MicroScale Thermophoresis demonstrated that the NSP12 P323L or P323L/G671S mutation stabilized the NSP12-NSP7-NSP8 complex interaction. Furthermore, reverse genetics-derived SARS-CoV-2 variants containing the NSP12 P323L or P323L/G671S mutation displayed enhanced replication at 33{degrees}C, and high transmission in ferrets. This suggests that the evolutionarily forced NSP12 P323L and P323L/G671S mutations of recent SARS-CoV-2 VOC strains are associated with increases of the RdRp complex stability and enzymatic activity, promoting the high transmissibility.

microbiology↗

bioRxiv2022

Transcription coactivator OCA-B/Pou2af1 is necessary and sufficient to promote T cell-intrinsic CD4 memory

The molecular mechanisms leading to the establishment of immunological memory are inadequately understood, limiting the development of effective vaccines and durable anti-tumor immune therapies. Here we show that ectopic OCA-B expression is sufficient to improve antiviral memory recall responses, while having minimal effects on primary effector responses. At peak viral response short lived effector T cell populations are expanded but show increased Gadd45b and Socs2 expression, while memory precursor effector cells show increased expression of Bcl2, Il7r and Tcf7 on a per-cell basis. Using an OCA-B mCherry reporter mouse line, we observe high OCA-B expression in CD4+ TCM cells. We show that early in viral infection, endogenously elevated OCA-B expression prospectively identifies memory precursor cells with increased survival capability and memory recall potential. Cumulatively, the results demonstrate that OCA-B is both necessary and sufficient to promote CD4 T cell memory in vivo and can be used to prospectively identify memory precursor cells. SignificanceCD4+ T cell memory is incompletely defined and memory progenitors difficult to identify. Here, we show that expression of the OCA-B transcription coactivator in CD4+ T cells is necessary and sufficient to drive productive memory. Using a novel mCherry reporter mouse line, we show that OCA-B expression enriches for responding effector cells with elevated memory potential. The results show that OCA-B expression in T cells is sufficient to promote CD4+ memory formation and marks memory precursor cells.

immunology↗

bioRxiv2022

Melanoma-intrinsic NR2F6 activity regulates anti-tumor immunity

Nuclear receptors (NRs) have been implicated in tumor and immune cell regulation, most notably in hormone-dependent cancers. Here we identify a tumor-intrinsic function of the orphan nuclear receptor NR2F6 in regulating anti-tumor immunity. We selected NR2F6 among 48 candidate NRs based on an expression pattern in melanoma patient specimens coinciding with the IFN{gamma} signature, which is associated with positive responses to immunotherapy and favorable patient outcomes. Further delay of melanoma development was achieved upon combination of NR2F6 inhibition with anti-PD1 therapy. NR2F6 loss in B16F10 and YUMM1.7 melanoma cells attenuated tumor development in immune-competent but not -incompetent mice via increased CD8+ T cell infiltration and activation. Inhibition of NACC1 and FKBP10, identified here as NR2F6 effectors, phenocopied NR2F6 loss. Remarkably, inoculation of NR2F6 KD melanoma cells into mice genetically deficient in NR2F6 further enhanced tumor growth inhibition. Identifying a tumor-intrinsic function of NR2F6 which complements its tumor-extrinsic role could be thus exploited to develop novel anti-cancer therapies.

cancer biology↗

bioRxiv2022

Style Transfer Generative Adversarial Networks to Harmonize Multi-Site MRI to a Single Reference Image to Avoid Over-Correction

Recent work within neuroimaging consortia have aimed to identify reproducible, and often subtle, brain signatures of psychiatric or neurological conditions. To allow for high-powered brain imaging analyses, it is often necessary to pool MR images that were acquired with different protocols across multiple scanners. Current retrospective harmonization techniques have shown promise in removing cross-site image variation. However, most statistical approaches may over-correct for technical, scanning-related, variation as they cannot distinguish between confounded image-acquisition based variability and cross-site population variability. Such statistical methods often require that datasets contain subjects or patient groups with similar clinical or demographic information to isolate the acquisition-based variability. To overcome this limitation, we consider cross-site MRI image harmonization as a style transfer problem rather than a domain transfer problem. Using a fully unsupervised deep-learning framework based on a generative adversarial network (GAN), we show that MR images can be harmonized by inserting the style information encoded from a single reference image, without knowing their site/scanner labels a priori. We trained our model using data from five large-scale multi-site datasets with varied demographics. Results demonstrated that our style-encoding model can harmonize MR images, and match intensity profiles, without relying on traveling subjects. This model also avoids the need to control for clinical, diagnostic, or demographic information. We highlight the effectiveness of our method for clinical research by comparing extracted cortical and subcortical features, brain-age estimates, and case-control effect sizes before and after the harmonization. We showed that our harmonization removed the cross-site variances, while preserving the anatomical information and clinical meaningful patterns. We further demonstrated that with a diverse training set, our method successfully harmonized MR images collected from unseen scanners and protocols, suggesting a promising novel tool for ongoing collaborative studies. Source code is released in USC-IGC/style_transfer_harmonization (github.com).

neuroscience↗

bioRxiv2022

Multiscale compression-induced restructuring of stacked lipid bilayers: from buckling delamination to molecular packing

Lipid membranes in nature adapt and reconfigure to changes in composition, temperature, humidity, and mechanics. For instance, the oscillating mechanical forces on lung cells and alveoli influence membrane synthesis and structure during breathing. However, despite advances in the understanding of lipid membrane phase behavior and mechanics of tissue, there is a critical knowledge gap regarding the response of lipid membranes to micromechanical forces. Most studies of lipid membrane mechanics use supported lipid bilayer systems missing the structural complexity of pulmonary lipids in alveolar membranes comprising multi-bilayer interconnected stacks. Here, we elucidate the collective response of the major component of pulmonary lipids to strain in the form of multi-bilayer stacks supported on flexible elastomer substrates. We utilize X-ray diffraction, scanning probe microscopy, confocal microscopy, and molecular dynamics simulation to show that lipid multilayered films both in gel and fluid states evolve structurally and mechanically in response to compression at multiple length scales. Specifically, compression leads to increased disorder of lipid alkyl chains comparable to the effect of cholesterol on gel phases as a direct result of the formation of nanoscale undulations in the lipid multilayers, also inducing buckling delamination and enhancing multi-bilayer alignment. We propose this cooperative short- and long-range reconfiguration of lipid multilayered films under compression constitutes a mechanism to accommodate stress and substrate topography. Our work raises fundamental insights regarding the adaptability of complex lipid membranes to mechanical stimuli. This is critical to several technologies requiring mechanically reconfigurable surfaces such as the development of electronic devices interfacing biological materials.

biophysics↗

bioRxiv2022

Working strokes produced by curling protofilaments at disassembling microtubule tips can be biochemically tuned and vary with species

The disassembly of microtubules can generate force and drive intracellular motility. During mitosis, chromosomes remain persistently attached via kinetochores to the tips of disassembling microtubules, which pull the sister chromatids apart. According to the conformational wave hypothesis, such force generation requires that protofilaments curl outward from the disassembling tips to exert pulling force directly on kinetochores. Rigorously testing this idea will require modifying the mechanical and energetic properties of curling protofilaments, but no way to do so has yet been described. Here, by direct measurement of working strokes generated in vitro by curling protofilaments, we show that their mechanical energy output can be increased by adding magnesium, and that yeast microtubules generate larger and more energetic working strokes than bovine microtubules. Both the magnesium and species-dependent increases in work output can be explained by a lengthening of protofilament curls, without any change in their bending stiffness or intrinsic curvature. These observations demonstrate how work output from curling protofilaments can be tuned and suggest evolutionary conservation of the amount of curvature strain energy stored in the microtubule lattice.

biophysics↗