Biology subjects

Akdogan, E.

Publications and source records attributed to Akdogan, E..

6 recordsLinked to original sources

bioRxiv2025

Parallel CRISPR screens reveal pathways controlling the cell surface levels of the attractant receptor FPR1

Levels of receptors on the cell surface control the sensitivity of immune cells, which is central to achieving effective responses while avoiding inflammatory diseases. The chemoattractant receptor FPR1 activates strong chemotactic and cytotoxic responses in neutrophils. While its precise regulation is critical for appropriate responses, the mechanisms controlling its cell surface levels remain unclear. Here, we investigated the roles of both classic and unknown regulators. First, we found that multiple G protein-coupled receptor kinases (GRK2, GRK3, and GRK6), and both {beta}-arrestin1 and 2 are important for FPR1 internalization. However, FPR1 uses multiple endocytic pathways, as cells lacking {beta}-arrestins have strong, but incomplete defects in FPR1 internalization. Moreover, we performed two parallel genome-wide CRISPR/Cas9 screens for the regulators of FPR1 surface expression and internalization in a neutrophil-like cell line. These screens identified regulators of FPR1 surface expression, recycling, and endocytosis. We identified the formin mDia1 and the small GTPase Arf6 as specific regulators of FPR1 internalization, and we confirmed these phenotypes using chemical inhibitors in primary human neutrophils. Furthermore, our data indicates that Arf6 contributes to the {beta}-arrestin-independent pathway. Collectively, our results clarify the contributions of GRKs and {beta}-arrestins in FPR1 internalization, indicate that internalization involves multiple compensatory routes, and uncover previously unidentified regulators of FPR1 biogenesis and trafficking, providing new mechanistic insight into these processes.

cell biology↗

bioRxiv2022

Cell migration CRISPRi screens in human neutrophils reveal regulators of context-dependent migration and differentiation state

Neutrophils are the most abundant leukocyte in humans and provide a critical early line of defense as part of our innate immune system. Their exquisite sensitivity to chemical gradients and ability to rapidly migrate make them especially suited to protect against infection. However, their terminal differentiation status and short lifetime (on the order of days) have hindered their study. Furthermore, while modern CRISPR-based gene perturbation strategies now allow comprehensive, genome-scale screens in human cells, their application to complex and dynamic processes like cell migration remain limited. Using HL-60 cells, a leukemia cell line that can be differentiated into neutrophil-like cells, we have developed multiple cell migration screen strategies that provide comprehensive, genome-wide discovery of molecular factors that are critical for directed (chemotaxis), undirected (chemokinesis), and 3D amoeboid cell migration in these fast-moving cells. Combining these assays with additional, pooled, genome-wide CRISPR interference dropout screens of cell proliferation and neutrophil differentiation, we have identified a comprehensive set of genes that are important across the processes of cellular growth, differentiation, and migration. This combined dataset highlights a particular reliance upon mTORC1 signaling that alters neutrophil lifetime, migration phenotype, and sensitivity to chemotactic cues. Across our cell migration screens, we identified several hundred genes important for migration including those with specific roles only in particular migratory contexts. This genome-wide screening strategy, therefore, provides an invaluable approach to the study of neutrophils and provides a resource that will inform future studies of cell migration in these and other rapidly migrating cells.

cell biology↗

bioRxiv2022

Phosphate Starvation Signaling Increases Mitochondrial Membrane Potential through Respiration-independent Mechanisms

Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both through inducing the electron transport chain and the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of the mitochondrial membrane potential is also observed in primary and immortalized mammalian cells as well as in Drosophila. These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial functions even with defective mitochondria.

cell biology↗

bioRxiv2022

Signaling dynamics distinguish high and low priority neutrophil attractant receptors

Human neutrophils respond to multiple chemical attractants to guide their migration from the vasculature to sites of infection and injury, where they clear pathogens and amplify inflammation. To properly focus their responses during this complex navigation, neutrophils distinguish between attractants to modulate their responses. They prioritize pathogen and injury derived signals over long-range inflammatory signals secreted by host cells. Different attractants can also drive qualitatively different modes of migration. As receptors recognizing both classes of attractant couple to Gi G-proteins, it remains unclear how downstream signaling pathways distinguish and prioritize between the inputs. Here, we use live-cell imaging to demonstrate that the responses differ in their signaling dynamics: low priority attractants cause transient responses, while responses to high priority attractants are sustained. We observe this difference in both primary neutrophils and differentiated HL-60 cells, for signaling outputs of calcium, a major regulator of secretion, and Cdc42, a primary regulator of polarity and cell steering. We find that the rapid attenuation of Cdc42 activation in response to LTB4 depends on the threonine 308 and serine 310 phosphorylation sites in the C-terminal tail of its receptor LTB4R, in a manner independent of endocytosis. Mutation of these residues to alanine impairs attractant prioritization, although it does not affect attractant-dependent differences in migration persistence. Our results indicate that distinct temporal regulation of shared signaling pathways distinguishes receptors and contributes to chemoattractant prioritization.

cell biology↗

bioRxiv2021

Optogenetic control of receptors reveals distinct roles for actin- and Cdc42-dependent negative signals in chemotactic signal processing

During chemotaxis, neutrophils use cell surface G-Protein Coupled Receptors (GPCRs) to detect chemoattractant gradients1-4. The downstream signaling system is wired with multiple feedback loops that amplify weak inputs and promote spatial separation of cell front and rear activities1, 5-8. Positive feedback could promote rapid signal spreading9, yet information from the receptors is transmitted with high spatial fidelity, enabling detection of small differences in chemoattractant concentration across the cell1. How the signal transduction network achieves signal amplification while preserving spatial information remains unclear. The GTPase Cdc42 is a cell-front polarity coordinator that is predictive of cell turning, suggesting an important role in spatial processing10. To directly measure information flow from receptors to Cdc42, we paired zebrafish parapinopsina, an optogenetic GPCR that allows reversible ON/OFF receptor control with a spectrally compatible red/far red Cdc42 FRET biosensor. Using this new toolkit, we show that positive and negative signals downstream of G-proteins shape a rapid, dose-dependent Cdc42 response. Furthermore, F-actin and Cdc42 itself provide two distinct negative signals that limit the duration and spatial spread of Cdc42 activation, maintaining output signals local to the originating receptors.

cell biology↗

bioRxiv2018

A bacteria-derived tail anchor localizes to peroxisomes in yeast and mammalian cells

Prokaryotes can provide new genetic information to eukaryotes by horizontal gene transfer (HGT), and such transfers are likely to have been particularly consequential at the dawn of eukaryogenesis. Since eukaryotes are highly compartmentalized, it is worthwhile to consider the mechanisms by which newly transferred proteins might reach diverse organellar destinations. Toward this goal, we have focused our attention upon the behavior of bacteria-derived tail anchors (TAs) expressed in the eukaryote Saccharomyces cerevisiae. In this study, we report that a predicted membrane-associated domain of the Escherichia coli YgiM protein is specifically trafficked to peroxisomes in budding yeast, can be found at a pre-peroxisomal compartment (PPC) upon disruption of peroxisomal biogenesis, and can functionally replace an endogenous peroxisome-directed TA. Furthermore, the YgiM(TA) can also localize to peroxisomes in mammalian cells. Since the YgiM(TA) plays no endogenous role in peroxisomal function or assembly, this domain is likely to serve as an excellent tool toward illumination of the mechanisms by which TAs can travel to peroxisomes. Moreover, our findings emphasize the ease with which bacteria-derived sequences might target to organelles in eukaryotic cells following HGT, and we discuss the importance of flexible recognition of organelle targeting information during and after eukaryogenesis.

evolutionary biology↗