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Results for “cell biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2023

Spindle architecture constrains karyotype in budding yeast

The eukaryotic cell division machinery must rapidly and reproducibly duplicate and partition the cells chromosomes in a carefully coordinated process. However, chromosome number varies dramatically between genomes, even on short evolutionary timescales. We sought to understand how the mitotic machinery senses and responds to karyotypic changes by using a series of budding yeast strains in which the native chromosomes have been successively fused. Using a combination of cell biological profiling, genetic engineering, and experimental evolution, we show that chromosome fusions are well tolerated up until a critical point. Cells with fewer than five centromeres lack the necessary number of kinetochore-microtubule attachments needed to counter outward forces in the metaphase spindle, triggering the spindle assembly checkpoint and prolonging metaphase. Our findings demonstrate that spindle architecture is a constraining factor for karyotype evolution.

cell biology↗

bioRxiv2024

Unbiased identification of cell identity in dense mixed neural cultures

Induced pluripotent stem cell (iPSC) technology is revolutionizing cell biology. However, the variability between individual iPSC lines and the lack of efficient technology to comprehensively characterize iPSC-derived cell types hinder its adoption in routine preclinical screening settings. To facilitate the validation of iPSC-derived cell culture composition, we have implemented an imaging assay based on cell painting and convolutional neural networks to recognize cell types in dense and mixed cultures with high fidelity. We have benchmarked our approach using pure and mixed cultures of neuroblastoma and astrocytoma cell lines and attained a classification accuracy above 96%. Through iterative data erosion we found that inputs containing the nuclear region of interest and its close environment, allow achieving equally high classification accuracy as inputs containing the whole cell for semi-confluent cultures and preserved prediction accuracy even in very dense cultures. We then applied this regionally restricted cell profiling approach to evaluate the differentiation status of iPSC-derived neural cultures, by determining the ratio of postmitotic neurons and neural progenitors. We found that the cell-based prediction significantly outperformed an approach in which the time in culture was used as classification criterion (96% vs. 86%, resp.). In mixed iPSC-derived neuronal cultures, microglia could be unequivocally discriminated from neurons, regardless of their reactivity state. A tiered strategy, allowed for discriminating microglial cell states as well, albeit with lower accuracy. Thus, morphological single cell profiling provides a means to quantify cell composition in complex mixed neural cultures and holds promise for use in quality control of iPSC-derived cell culture models.

cell biology↗

bioRxiv2026

Unbranched SPIN90-Arp2/3 actin promotes stress fiber speed and focal adhesion maturation

The Arp2/3 complex has long been considered to only assemble branched actin structures in the cell (lamellipodia, endocytic patches, comet tails, and many more). We show for the first time by single-molecule tracking (SMT) that the Arp2/3 complex and SPIN90, which activates Arp2/3 complex to nucleate unbranched filaments, bind to and move in the basal cortex with stress fibers and focal adhesions (FA) that, unlike known sites of Arp2/3 enrichment, employ linear actin bundles. SPIN90 knockout in U2OS cells significantly increases the rate of collective cell migration while decreasing cellular traction (myosin-II and actin speeds) and adhesion (FA size and maturation markers). SPIN90s SH3 domain, similar to its adapter protein Nck1, shows enrichment in FAs, suggesting a possible location for SPIN90-Arp2/3 complex activity. Together, our findings indicate that SPIN90-Arp2/3 nucleated filaments also function in stress fibers where they help define the mechanics of traction and adhesion to regulate cell motility. Significance statementThe Arp2/3 complex, the branched actin nucleator, drives pushing forces in the cytoplasm, yet its diffuse cortical localization has obscured its functions within unbranched structures. In conventional microscopy, associations of molecules with specific structures are impossible to verify when there is little enrichment over the cytoplasm. Whereas other methods fail to provide convincing evidence of such associations, we used single molecule tracking in live cells to identify molecules that track the motions of stress fibers. Thus, we were able to discover that the Arp2/3 complex and SPIN90, proteins whose roles in cell biology have been thus far limited to branched actin networks, directly integrate within the unbranched stress fiber network to promote cell adhesion and traction for cell migration.

cell biology↗

bioRxiv2022

2-hydroxyglutarate controls centromere and heterochromatin conformation and function in the male germline

2-hydroxyglutarate (2HG) is recognized as an epigenetic regulator in cancer and some transient biological processes. Of all organs, the testis harbors the highest baseline physiological levels of 2HG, yet its putative functions in germ cell biology are unknown. Here we show that 2HG is generated in specific stages of spermatogenesis by the testis specific lactate dehydrogenase C (LDHC), beginning at the last stages of prophase I. Unexpectedly LDHC enters nuclei and concentrates in centromeres. LDHC-generated L-2HG controls centromere condensation and pericentromeric heterochromatin organization through multiple effects including clustering of chromocenters, centromere and chromocenter condensation and expression of satellite RNAs. These effects are rapid and specific to L but not D-2HG. In vivo depletion of L-2HG causes centromere malfunction and activation of the spindle assembly checkpoint. Our findings reveal that 2HG can directly affect centromere and pericentromeric heterochromatin conformation and function and is necessary for licensing chromosome segregation.

cell biology↗

bioRxiv2022

Increased Relative Biological Effectiveness of Orthovoltage X-rays Compared to γ-rays in Preclinical Irradiation

PurposeRadionuclide irradiators (137Cs and 60Co) are commonly used in preclinical studies ranging from cancer therapy to stem cell biology. There are institutional initiatives to replace radionuclide sources with lower-energy X-ray sources amidst concerns of radiological terrorism. As researchers transition, there are questions whether the biological effects of {gamma}-rays may be recapitulated with orthovoltage X-rays, since different energy may cause different biological effects. We, therefore, sought to compare the effects of orthovoltage X-rays and 137Cs {gamma}-rays using mouse models of acute radiation syndrome. Experimental Design137Cs {gamma}-rays were compared with Orthovoltage X-rays, generated at 300 kVp, 10 mA with 1 mm Cu or Thoraeus filtration. We assessed 30-day overall survival following whole-body irradiation and calculated LD50 by logistic regression. Comparing equivalent doses delivered with different average energies ([E]), we assessed bone marrow, spleen, and intestinal histology and flow cytometry. ResultsThe LD50 doses are 6.7 Gy, 7.4 Gy and 8.1 Gy with 1 mm Cu filtered ([E]=120 keV), and Thoraeus filtered X-rays ([E]=160 keV), and 137Cs (E=662 keV), respectively. At constant dose, hematopoietic injury was most severe with 1 mm Cu filtered X-rays with the greatest reduction in bone marrow cellularity, stem and progenitor populations, and intestinal crypts and OLFM4+ intestinal stem cells. Thoraeus filtered X-rays provoked an intermediate phenotype, with 137Cs showing the least damage. ConclusionsOur study reveals a dichotomy between physical dose and biological effect relevant as researchers transition to orthovoltage X-rays. With decreasing energy, there is increasing hematopoietic and intestinal injury, necessitating dose-reduction to achieve comparable biological effects. Statement of Translational RelevanceRadiation is used in translational studies in fields ranging from hematopoiesis and stem cell biology to cancer radiotherapy, with 137Cs and 60Co radionuclide sources serving as the most common irradiators. Due to the threat of radiological terrorism using stolen radionuclides, there are institutional initiatives to replace these sources with orthovoltage X-ray irradiators. Yet, as shown in this study, the biological effects of radiation are highly dependent on radiation energy. Lower energy orthovoltage X-rays are absorbed differently than higher energy radionuclide {gamma}-rays, provoking more severe hematopoietic, immunologic, and gastrointestinal radiation injury. Thus, an identical physical dose delivered with beams of differing energy does not produce the same biologic effect. As researchers transition between these sources, it is critical that we appreciate that radiation doses are not interchangeable between them. Understanding the significance of physical dose delivered using different methods will allow us to contextualize past results with future studies.

pathology↗

bioRxiv2017

The tumor suppressor APC is an attenuator of spindle-pulling forces during C. elegans asymmetric cell division

The adenomatous polyposis coli (APC) tumor suppressor has dual functions in Wnt/{beta}-catenin signaling and accurate chromosome segregation, and is frequently mutated in colorectal cancers. Although APC contributes to proper cell division, the underlying mechanisms remain poorly understood. Here we show that C. elegans APR-1/APC is an attenuator of the pulling forces acting on the mitotic spindle. During asymmetric cell division of the C. elegans zygote, a LIN-5/NuMA protein complex localizes dynein to the cell cortex to generate pulling forces on astral microtubules that position the mitotic spindle. We found that APR-1 localizes to the anterior cell cortex in a Par-aPKC polarity-dependent manner and suppresses anterior centrosome movements. Our combined cell biological and mathematical analyses support the conclusion that cortical APR-1 reduces force generation by stabilizing microtubule plus ends at the cell cortex. Furthermore, APR-1 functions in coordination with LIN-5 phosphorylation to attenuate spindle pulling forces. Our results document a physical basis for spindle-pulling force attenuation, which may be generally used in asymmetric cell division, and when disrupted potentially contributes to division defects in cancer.\n\nSignificance StatementAPC (adenomatous polyposis coli) is a Wnt signaling component as well as a microtubule-associated protein, and its mutations are frequently associated with colorectal cancers in humans. Although APC stabilizes microtubules (MTs), its mechanical role during cell division is largely unknown. Here we show that APC is an attenuator of forces acting on the mitotic spindle during asymmetric cell division of the C. elegans zygote. We performed live-imaging, laser-microsurgery, and numerical simulation to show how APC suppresses spindle pulling force generation by stabilizing microtubule plus-ends and reducing microtubule catastrophe frequency at the cell cortex. Our study is the first to document a mechanical role for the APC protein, and provides a physical basis for spindle-pulling force attenuation.

cell biology↗

bioRxiv2017

Assay to visualize specific protein oxidation reveals spatio-temporal regulation of SHP2

Reactive oxygen species (ROS) are produced transiently in response to cell stimuli, and function as second messengers that oxidize target proteins. Protein-tyrosine phosphatases (PTPs) are important ROS targets, whose oxidation results in rapid, reversible, catalytic inactivation. Despite increasing evidence for the importance of PTP oxidation in signal transduction, the cell biological details of ROS-catalyzed PTP inactivation have remained largely unclear, due to our inability to visualize PTP oxidation in cells. By combining proximity ligation assay (PLA) with chemical labeling of cysteine residues in the sulfenic acid state, we visualize oxidized Src homology 2 domain-containing protein-tyrosine phosphatase 2 (SHP2). We find that platelet-derived growth factor (PDGF) evokes transient oxidation on or close to RAB5+/EEA1-endosomes. SHP2 oxidation requires NADPH oxidases (NOXs), and oxidized SHP2 co-localizes with PDGF receptor and NOX1/4. Our data demonstrate spatially and temporally limited protein oxidation within cells, and suggest that PDGF-dependent \"redoxosomes,\" contribute to proper signal transduction.

cell biology↗

bioRxiv2021

Scaling of subcellular structures with cell length through decelerated growth

How cells tune the size of their subcellular parts to scale with cell size is a fundamental question in cell biology. Until now, most studies on the size control of organelles and other subcellular structures have focused on scaling relationships with cell volume, which can be explained by limiting pool mechanisms. Here, we uncover a distinct scaling relationship with cell length rather than volume, revealed by mathematical modeling and quantitative imaging of yeast actin cables. The extension rate of cables decelerates as they approach the rear of the cell, until cable length matches cell length. Further, the deceleration rate scales with cell length. These observations reveal a new mode of scaling that senses the linear dimensions of a cell. One Sentence SummaryAs actin cables in yeast cells grow longer, their extension rate decelerates, enabling the cable length to match cell length.

cell biology↗

bioRxiv2021

The Tabula Sapiens: a single cell transcriptomic atlas of multiple organs from individual human donors

Molecular characterization of cell types using single cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues and tissue specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, the tissue specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type specific RNA splicing was discovered and analyzed across tissues within an individual.

cell biology↗

bioRxiv2023

Streamlined intravital imaging approach for long-term monitoring of epithelial cell dynamics on an inverted confocal microscope.

Understanding normal and aberrant in vivo cell behaviors is necessary to develop clinical interventions to thwart disease initiation and progression. It is therefore critical to optimize imaging approaches that facilitate the observation of cell dynamics in situ, where tissue structure and composition remains unperturbed. The epidermis is the bodys outermost barrier as well as the source of the most prevalent human cancers, namely cutaneous skin carcinomas. The accessibility of skin tissue presents a unique opportunity to monitor epithelial and dermal cell behaviors in intact animals using noninvasive intravital microscopy. Nevertheless, this sophisticated imaging approach has primarily been achieved using upright multiphoton microscopy, which represents a significant barrier-for-entry for most investigators. In this study, we present a custom-designed 3D-printed microscope stage insert suitable for use with inverted confocal microscopes that streamlines long-term intravital imaging of ear skin in live transgenic mice. We believe this invention, which may be readily customized to image other organ systems, will prove invaluable to the cell biology community by significantly enhancing the accessibility of intravital microscopy. This technological advancement is critical to bolster our understanding of live cell dynamics in both normal and disease contexts.

cell biology↗

bioRxiv2023

MuSK-BMP signaling in adult muscle stem cells maintains quiescence and regulates myofiber size

A central question in adult stem cell biology is elucidating the signaling pathways regulating their dynamics and function in diverse physiological and age-related contexts. Muscle stem cells in adults (Satellite Cells; SCs) are generally quiescent but can activate and contribute to muscle repair and growth. Here we tested the role of the MuSK-BMP pathway in regulating adult SC quiescence by deletion of the BMP-binding MuSK Ig3 domain ( {Delta}Ig3-MuSK). At 3 months of age SC and myonuclei numbers and myofiber size were comparable to WT. However, at 5 months of age SC density was decreased while myofiber size, myonuclear number and grip strength were increased - indicating that SCs had activated and productively fused into the myofibers over this interval. Transcriptomic analysis showed that SCs from uninjured {Delta}Ig3-MuSK mice exhibit signatures of activation. Regeneration experiments showed that {Delta}Ig3-MuSK SCs maintain full stem cell function. Expression of {Delta}Ig3-MuSK in adult SCs was sufficient to break quiescence and increase myofiber size. We conclude that the MuSK-BMP pathway regulates SC quiescence and myofiber size in a cell autonomous, age-dependent manner. Targeting MuSK-BMP signaling in muscle stem cells thus emerges a therapeutic strategy for promoting muscle growth and function in the settings of injury, disease, and aging. HighlightsO_LIMuSK, in its role as a BMP co-receptor, regulates adult muscle stem cell quiescence C_LIO_LIThe MuSK-BMP pathway acts cell autonomously C_LIO_LIIncreased muscle size and function with preservation of myonuclear density and stemness in mice with attenuated MuSK-BMP signaling C_LI

cell biology↗

bioRxiv2024

A first-principles geometric model for dynamics of motor-driven centrosomal asters

The centrosomal aster is a mobile and adaptable cellular organelle that exerts and transmits forces necessary for tasks such as nuclear migration and spindle positioning. Recent experimental and theoretical studies of nematode and human cells demonstrate that pulling forces on asters by cortically anchored force generators are dominant during such processes. Here we present a comprehensive investigation of a first-principles model of aster dynamics, the S-model (S for stoichiometry), based solely on such forces. The model evolves the astral centrosome position, a probability field of cell-surface motor occupancy by centrosomal microtubules (under an assumption of stoichiometric binding), and free boundaries of unattached, growing microtubules. We show how cell shape affects the stability of centering of the aster, and its transition to oscillations with increasing motor number. Seeking to understand observations in single-cell nematode embryos, we use highly accurate simulations to examine the nonlinear structures of the bifurcations, and demonstrate the importance of binding domain overlap to interpreting genetic perturbation experiments. We find a generally rich dynamical landscape, dependent upon cell shape, such as internal constant-velocity equatorial orbits of asters that can be seen as traveling wave solutions. Finally, we study the interactions of multiple asters which we demonstrate an effective mutual repulsion due to their competition for surface force generators. We find, amazingly, that centrosomes can relax onto the vertices of platonic and non-platonic solids, very closely mirroring the results of the classical Thomson problem for energy-minimizing configurations of electrons constrained to a sphere and interacting via repulsive Coulomb potentials. Our findings both explain experimental observations, providing insights into the mechanisms governing spindle positioning and cell division dynamics, and show the possibility of new nonlinear phenomena in cell biology.

cell biology↗

bioRxiv2024

Beta-hairpin Mechanism of Autoinhibition and Activation in the Kinesin-2 Family

Members of the kinesin-2 family coordinate with other motors to power diverse physiological processes, but the structural mechanisms regulating kinesin-2 activity have been unknown. Distinctively, kinesin-2s canonically function as heterotrimers of two different motor subunits (e.g. Kif3A and Kif3B) and Kap3, but the role of heterotrimerization has yet to fully emerge. Here, we combine structural, cell biological, and single-molecule approaches to dissect kinesin-2 regulation as a heterodimer, heterotrimer, and quaternary complex with a cargo adaptor (APC). We identify a conserved motif in the tail of kinesin-2s (the beta-hairpin motif) that controls kinesin-2 motility by sequestering the motor domains away from their microtubule track, using striking molecular mimicry. Our data reveal how Kap3 binds via a multipartite interface with Kif3A and Kif3B, and - rather than activating motility directly - provides a platform on which cargo adaptors can engage and occlude the beta-hairpin motif. Together, these data articulate a structural framework for kinesin-2 activation, coordination with dynein, and adaptation for different biological functions.

cell biology↗

bioRxiv2025

A conserved Hsp70 phosphorylation regulates cell cycle progression after DNA damage

Hsp70s are essential molecular chaperones that are increasingly recognized to be regulated by post-translational modifications. Here, we show that phosphorylation of a conserved threonine (T495), previously shown to be exploited by a Legionella pneumophila kinase to inhibit Hsp70, occurs endogenously in human cells in response to DNA damage, particularly when base excision repair is overburdened. This modification is cell cycle dependent, and in yeast, phosphomimetic or phosphonull Hsp70 variants disrupt G1/S progression under normal and DNA-damaging conditions. Biochemically, the phosphomimetic T495E mutation locks Hsp70 in an open-like conformation without blocking substrate engagement. Together, our results reveal a conserved mechanism by which dynamic Hsp70 phosphorylation regulates the G1/S transition, and delays cell cycle progression during DNA damage, highlighting how pathogen-derived insights can uncover fundamental cell biology principles.

cell biology↗

bioRxiv2018

Chromatin Condensation Fluctuations Rather Than Steady-State Predict Chromatin Accessibility

Chromatin accessibility to protein factors is critical for genome activities. Dynamic changes in nucleosomal DNA compaction and higher order chromatin structures are expected to allow specific sites to be accessible to regulatory factors and the transcriptional machinery. However, the dynamic properties of chromatin that regulate its accessibility are poorly understood. Here, we took advantage of the microenvironment sensitivity of the fluorescence lifetime of EGFP-H4 histone incorporated in chromatin to map in the nucleus of live cells the dynamics of chromatin condensation and its direct interaction with a tail acetylation recognition domain (the double bromodomain module of human TAFII250, dBD). We reveal chromatin condensation fluctuations supported by mechanisms fundamentally distinct from that of condensation. Fluctuations are spontaneous, yet their amplitudes are affected by their sub-nuclear localization and by distinct and competing mechanisms dependent on histone acetylation, ATP, and both. Moreover, we show that accessibility of acetylated histone H4 to dBD is not restricted by chromatin condensation nor predicted by acetylation, rather, it is predicted by chromatin condensation fluctuations.\n\nSignificanceIn higher eukaryotes, the structure and compaction of chromatin are considered as barriers to genome activities. Epigenetic marks such as post-translational modifications of histones can modify the structure and compaction of chromatin. The accessibility of protein factors to these epigenetic marks is therefore of paramount importance for genome activities. We reveal chromatin condensation fluctuations supported by mechanisms fundamentally distinct from that of condensation itself. We show that accessibility of acetylated histone H4 to double bromodomains is not restricted by chromatin condensation nor predicted by acetylation, rather, it is predicted by chromatin condensation fluctuations.\n\nClassificationBiological Sciences, Cell Biology

cell biology↗

bioRxiv2026

Preservation of Human Colonic Stem Cells Requires an ERK Dynamics Checkpoint Mediated by AKT

Colonic stem cells reside in a microenvironment enriched in epidermal growth factor, which is essential for their survival and can activate both PI3K-AKT and MAPK-ERK pathways. This predicts co-activation of both pathways within the growth factor-high stem cell compartment at the base of crypts. However, in patient-derived human colonic organoids and normal human tissue, stem cells maintain robust AKT activity while suppressing ERK signaling despite active EGFR engagement. As stem cells differentiate, they activate pulsatile ERK signaling, which is essential for migration, survival, and maintenance of barrier function. We show that AKT-dependent phosphorylation of RAF-1 at serine 259 establishes a post-receptor checkpoint that maintains ERK temporal dynamics in stem cells. Acute activation of ERK in stem cells triggers rapid global differentiation. Disruption of the ERK checkpoint via mutation of serine 259 leads to sustained AKT and ERK co-activation in stem cells. Unlike ERK/AKT coactivation driven by apoptosis, co-activation in the stem cell compartment results in the emergence of a neoplastic, architecturally disorganized cell population dominating the cell fate profile. Incredibly, introducing brief ERK pulses through AKT inhibition or ERK activation triggers re-differentiation of neoplastic cells. Consistent with duration-dependent MAPK encoding principles, these data demonstrate that regardless of baseline signaling amplitude, ERK signaling dynamics are epistatic to total kinase signaling load in human colonic stem cells. SIGNIFICANCEStem cells must balance self-renewal and differentiation while remaining responsive to continuous mitogenic stimulation to preserve tissue homeostasis. When self-renewal is impaired, wound healing and barrier integrity decline, whereas loss of proper differentiation drives tumorigenesis. Our findings demonstrate that this balance in the human colon is achieved through temporal control of kinase signaling rather than modulation of ligand availability. By establishing an AKT-dependent ERK dynamics checkpoint, colonic stem cells suppress differentiation-inducing ERK pulses while maintaining growth factor responsiveness. These results identify kinase dynamics as a fundamental determinant of epithelial homeostasis and suggest that subtle alterations in these dynamics may destabilize tissue organization during regeneration or chronic inflammation. Temporal encoding of kinase activity thus represents a central organizing principle in human stem cell biology.

cell biology↗

bioRxiv2021

Proximity interactome of LC3B in normal growth conditions

LC3 (Light Chain 3) is a key player of autophagy, a major stress-responsive proteolysis pathway promoting cellular homeostasis. It coordinates the formation and maturation of autophagosomes and recruits cargo to be further degraded upon autophagosome-lysosome fusion. To orchestrate its functions, LC3 binds to multiple proteins from the autophagosomes inner and outer membranes, but the full extent of these interactions is not known. Moreover, LC3 has been increasingly reported in other cellular locations than the autophagosome, with cellular outcome not fully understood and not all related to autophagy. Furthermore, novel functions of LC3 as well as autophagy can occur in cells growing in a normal medium thus in non-stressed conditions. A better knowledge of the molecule in proximity to LC3 in normal growth conditions will improve the understanding of LC3 function in autophagy and in other cell biology function. Using an APEX2 based proteomic approach, we have detected 407 proteins in proximity to the well-characterised LC3B isoform in non-stress conditions. These include known and novel LC3B proximity proteins, associated with various cell localisation and biological functions. Sixty-nine of these proteins contain a putative LIR (LC3 Interacting Region) including 41 not reported associated to autophagy. Several APEX2 hits were validated by co-immunoprecipitation and co-immunofluorescence. This study uncovers the LC3B global interactome and reveals novel LC3B interactors, irrespective of LC3B localisation and function. This knowledge could be exploited to better understand the role of LC3B in autophagy and non-autophagy cellular processes.

cell biology↗

bioRxiv2020

Efficient Generation of Isogenic Primary Human Myeloid Cells using CRISPR-Cas9 Ribonucleoproteins

Genome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14+ human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically-edited cells that retain critical markers of both myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection more than fifty-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate development of novel myeloid cellular therapies.

genetics↗