Biology subjects

Ciftci, D.

Publications and source records attributed to Ciftci, D..

5 recordsLinked to original sources

bioRxiv2026

Multimodal dynamics control activity of a glial glutamate transporter

Membrane transporters move polar solutes across lipid bilayers to regulate cellular metabolism, signaling, and drug distribution. These proteins operate via an alternating-access mechanism, cycling between extracellular-, intermediate-, and intracellular-facing conformations. The human excitatory amino acid transporter 1 (EAAT1) protects neurons from excitotoxic damage by mediating the uptake of glutamate and aspartate into glial cells. Defects in EAAT1 function result in numerous pathologies, including epilepsy and ataxia, suggesting that positive modulation of these transporters might ameliorate glutamate neurotoxicity. However, developing EAAT1 activators requires understanding the timing of conformational changes, which remain largely unexplored. Here, we establish an experimental platform that combines single-molecule Forster resonance energy transfer (smFRET) to monitor real-time conformational dynamics, single-transporter activity assays to correlate dynamics with function, and cryogenic electron microscopy (cryoEM) to visualize discrete conformations at high resolution. This platform enables detection of [A]ngstrom-scale movements of single transporter molecules in real time, revealing that EAAT1 intersperses rapid conformational dynamics with long pauses. Slow and fast dynamics can be modulated by substrates, membrane composition, and mutations, and are correlated with the enrichment of specific structural states. We leverage this platform to investigate an EAAT1 mutation associated with severe episodic ataxia and show that it inhibits transport by stabilizing a paused cytoplasm-facing conformation. These results identify multimodal dynamics as an intrinsic, regulatable feature of EAAT1 function and, therefore, a potential therapeutic target. Henceforth, our integrated platform will facilitate investigations of other regulatory factors, including the effects of small-molecule and lipid modulators on the transport cycle.

biophysics↗

bioRxiv2026

Comprehensive Lineage Tracing Maps the Landscape of Cell Fate Decisions in Mouse Embryogenesis

A comprehensive cell fate map of mammalian embryogenesis has remained out of reach due to the scale, cellular diversity, and non-deterministic nature of development in utero. Here, we use PEtracer to continuously install heritable genetic marks as cells divide, reconstructing lineage trees that resolve [~]75% of cell divisions across >1.5 million cells from 16 mouse embryos collected at half-day intervals from E7.5-E10.0. We pair these trees with deep transcriptional profiling to chart the landscape of cell fate decisions during gastrulation and early organogenesis. Using these data, we quantify cell fate biases, restriction timing, progenitor pool sizes, and lineage relationships across the embryo, revealing strikingly reproducible lineage architecture across replicate embryos despite the regulative flexibility of mammalian development. We further show how lineage, spatial position, and signaling jointly determine fate outcomes and timing, with their relative influence varying by tissue. This dataset provides a quantitative framework for understanding cell fate specification and a lineage-resolved reference for generating and contextualizing developmental hypotheses at organismal scale.

developmental biology↗

bioRxiv2024

Multiplexed optical barcoding and sequencing for spatial omics

Spatial omics has brought a fundamental change in the way that we study cell and tissue biology in health and disease. Among various spatial omics methods, genome-scale imaging allows transcriptomic, 3D-genomic, and epigenomic profiling of individual cells with high spatial (subcellular) resolution but often requires a preselection of targeted genes or genomic loci. On the other hand, spatially dependent barcoding of molecules followed by sequencing provides untargeted, genome-wide profiling but typically lacks single-cell resolution. Here, we report a spatial omics method that could potentially combine the power of the two approaches by optically controlled spatial barcoding followed by sequencing. Specifically, we utilize patterned light to encode the locations of cells in tissues using oligonucleotide-based barcodes and then identify the barcoded molecular content, such as mRNAs, by sequencing. This optical barcoding method is designed with multiplexing and error-correction capacity and achieved by a light-directed ligation chemistry that attaches distinct nucleic-acid sequences to the reverse transcribed cDNA products at different locations. As a proof of principle for this method, we demonstrated high-efficiency in situ light-directed ligation, spatially dependent barcoding with multiplexed light-controlled ligations, and high-accuracy detection of spatially barcoded mRNAs in cells.

genomics↗

bioRxiv2021

Heterogeneous substrate binding in a glutamate transporter homologue

Integral membrane glutamate transporters couple the concentrative substrate transport to ion gradients. There is a wealth of structural and mechanistic information about this family. Recent studies revealed transport rate heterogeneity in an archaeal homologue GltPh, inconsistent with simple kinetic models, but its structural and mechanistic determinants remain undefined. In a mutant GltPh, which exclusively populates the outward-facing state, we demonstrate the co-existence of at least two sub-states in slow equilibrium binding the substrate with different apparent affinities. Wild-type GltPh shows similar binding properties, and modulation of the sub-state equilibrium correlates with transport rates. Following binding, the low-affinity sub-state of the mutant is transient. Consistently, cryo-EM on samples frozen within seconds after substrate addition reveals the presence of structural classes with perturbed helical packing of the extracellular half of the transport domain in regions adjacent to the binding site. In contrast, an equilibrated structure does not show such classes. The structure at 2.2 [A] resolution details a pattern of waters in the intracellular half of the domain and resolves classes with subtle differences in the substrate-binding site. We hypothesize that the rigid cytoplasmic half of the domain mediates substrate and ion recognition and coupling, while the extracellular labile half sets the affinity and dynamic properties.

biophysics↗

bioRxiv2020

The high-energy transition state of a membrane transporter

Membrane transporters mediate cellular uptake of nutrients, signaling molecules and drugs. Their overall mechanisms are often well understood, but the structural features setting their rates are mostly unknown. Earlier single-molecule fluorescence imaging of a model glutamate transporter homologue suggested that the slow conformational transition from the outward- to the inward-facing state, when the bound substrate is translocated from the extracellular to the cytoplasmic side of the membrane, is rate-limiting to transport. Here, we aim to gain insight into the structure of the high-energy transition state that limits the rate of this critical isomerization reaction. Using bioinformatics, we identify gain-of-function mutants of the transporter and apply linear free energy relationship analysis to infer that the transition state structurally resembles the inward-facing conformation. Based on these analyses, we propose an approach for allosteric modulation of these transporters.

biophysics↗