Biology subjects

Sykes, C.

Publications and source records attributed to Sykes, C..

8 recordsLinked to original sources

bioRxiv2026

Histone H3 availability is more important for development than H3.2 versus H3.3 subtype identity

The distinct contributions of replication-dependent and replication-independent histones to development and genome function remain unclear. In this study, we investigate how the distinct protein identities of the histone H3.2 and H3.3 subtypes contribute to development and gene regulation in Drosophila. Comparing animals in which the replication-independent H3.3 genes were mutated to produce the replication-dependent H3.2 protein with those carrying deletions of the replication-independent H3.3 genes revealed that replication-independent H3.3 is essential for fertility, adult locomotor behavior, and normal longevity. However, development to adulthood does not depend on which replication-independent H3 subtype is expressed from the H3.3 loci. Moreover, replication-independent H3.3 is not required to establish or maintain global patterns of chromatin accessibility or gene expression in the adult brain. Surprisingly, we find that expression of H3.2 from the replication-dependent HisC locus is essential in post-replicative cells in the absence of replication-independent H3.3, and we uncover a critical role for the HIRA histone chaperone complex in preserving genome function when replication-independent H3.3 is deleted. We conclude that an available pool of H3 is more critical than the specific identity of H3 in the pool.

genetics↗

bioRxiv2024

The nuclear lamin network passively responds to both active or passive cell movement through confinements

Physical models of cell motility rely mostly on cytoskeletal dynamical assembly. However, when cells move through the complex 3D environment of living tissues, they have to squeeze their nucleus that is stiffer than the rest of the cell. The lamin network, organised as a shell right underneath the nuclear membrane, contributes to the nuclear integrity and stiffness. Yet, its response during squeezed cell motility has never been fully characterised. As a result, up to now, the interpretations on the lamin response mechanism are mainly speculative. Here, we quantitatively map the lamin A/C distribution in both a microfluidic migration device and a microfluidic aspiration device. In the first case, the cell is actively involved in translocating the nucleus through the constriction, while in the second case, the cell behaves as a passive object that is pushed through the constriction by an external pressure. Using a quantitative description of the lamin shell response based on mass conservation arguments applied on the fluorescence signal of lamin, we show that in both cases of migration and aspiration, the response of the lamin network is passive. In this way, our results not only further elucidate the lamin response mechanism, but also allow to distinguish this passive deformation response from other active responses that may occur when the nucleus is squeezed through constrictions.

biophysics↗

bioRxiv2020

Validation of an LC-MS/MS Assay for the Simultaneous Determination of Bictegravir, Doravirine, and Raltegravir in Human Plasma

Bictegravir (BIC), an integrase inhibitor, and doravirine (DOR), a non-nucleoside reverse transcriptase inhibitor, were recently approved by the US FDA for HIV treatment and are recommended first line treatment options. Because certain clinical scenarios warrant using them in combination, we developed a fully validated LC-MS/MS method for simultaneous measurement of BIC and DOR, along with a legacy integrase inhibitor, raltegravir (RAL), in human plasma over a clinically relevant 1000-fold range for each analyte. These analytes were extracted from the plasma by protein precipitation with their stable, isotopically labeled internal standards (BIC-d5, 13C6-DOR, and RAL-d6). Following extraction, samples were analyzed by reverse phase chromatography on a Waters Atlantis T3 C18 (50×2.1mm, 3um particle size) column with subsequent detection by electrospray ionization in positive ion mode on an AB Sciex API-5000 triple quadrupole mass spectrometer. The assay was linear (R2>0.994) over the selected calibration ranges (20.0-20,000ng/mL (BIC), 3.00-3,000ng/mL (DOR), and 10.0-10,000 (RAL)). The assay was accurate (inter-assay %Bias ≤±8.5) and precise (inter-assay %CV ≤11.4). This method was validated according to FDA guidance for industry and can be used to assess the pharmacokinetics of two newly approved antiretrovirals, or to support therapeutic drug monitoring for modern antiretroviral therapy.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗

bioRxiv2019

Actin shells control buckling and wrinkling of biomembranes

Global changes of cell shape under mechanical or osmotic external stresses are mostly controlled by the mechanics of the cortical actin cytoskeleton underlying the cell membrane. Some aspects of this process can be recapitulated in vitro on reconstituted actin-and-membrane systems. In this paper, we investigate how the mechanical properties of a branched actin network shell, polymerized at the surface of a liposome, control membrane shape when the volume is reduced. We observe a variety of membrane shapes depending on the actin thickness. Thin shells undergo buckling, characterized by a cup-shape deformation of the membrane that coincides with the one of the actin network. Thick shells produce membrane wrinkles, but do not deform their outer layer. For intermediate micrometerthick shells, wrinkling of the membrane is observed, and the actin layer is slightly deformed. Confronting our experimental results with a theoretical description, we determine the transition between buckling and wrinkling depending on the thickness of the actin shell and the size of the liposome. We thus unveil the generic mechanism by which biomembranes are able to accommodate their shape against mechanical compression, through thickness adaptation of their cortical cytoskeleton.

biophysics↗

bioRxiv2019

LINC complexes are mechanotransducers that discriminate Epithelial-Mesenchymal Transition programs

LINC complexes are transmembrane protein assemblies that physically connect the nucleo- and cytoskeletons through the nuclear envelope. Dysfunctions of LINC complexes are associated with pathologies such as cancer and muscular disorders. The mechanical roles of LINC complexes in these contexts are poorly understood. To address this, we used genetically encoded FRET biosensors of molecular tension in LINC complex proteins of fibroblastic and epithelial cells in culture. We exposed cells to mechanical, genetic and pharmacological perturbations, mimicking a range of physiological and pathological situations. We show that LINC complex proteins experience tension generated by the cytoskeleton and act as mechanical sensors of cell packing. Moreover, the LINC complex discriminates between inductions of partial and complete epithelial-mesenchymal transitions (EMT). We identify the implicated mechanisms, which associate nesprin tension sensing with -catenin capture at the nuclear envelope, thereby regulating {beta}-catenin transcription. Our data thus implicate that LINC complexes are mechanotransducers that fine-tune {beta}-catenin signaling in a manner dependent on the Epithelial-Mesenchymal Transition program.

cell biology↗

bioRxiv2019

Actin accumulates nesprin-2 at the front of the nucleus during confined cell migration

The mechanisms by which cells exert forces on their nuclei to migrate through openings smaller than the nuclear diameter remain unclear. In microfluidic devices, the hourglass shape of the nucleus and its strain patterns as it translocates through narrow constrictions suggest pulling forces. We use CRISPR/Cas9 to fluorescently label nesprin-2 giant, a protein that links the cytoskeleton to the interior of the nucleus. We demonstrate that nesprin-2 giant accumulates at the front of the nucleus during nuclear deformation through narrow constrictions, independently of the nuclear lamina. We find that nesprins are more mobile than lamin A/C, at time scales similar to that of the accumulation. Using artificial constructs, we show that the actin-binding domain of nesprin-2 is necessary and sufficient to generate this accumulation, and that microtubules are not necessary. Actin filaments are organized in a barrel structure around the moving nucleus in the direction of movement, suggesting that this structure is responsible for redistribution of nesprins towards the front of the nucleus. Two-photon ablation and the use of drugs inhibiting the cytoskeleton demonstrate a pulling force on the nucleus from the front of the cell that is dependent on formin and actomyosin contractility. This elastic recoil is significantly reduced when nesprins are reduced at the nuclear envelope. We thus show that actin redistributes nesprin-2 giant towards the front of the nucleus and contributes to pulling the nucleus through narrow constrictions, in concert with myosin.

cell biology↗

bioRxiv2019

Actin modulates shape and mechanics of tubular membranes

The actin cytoskeleton shapes cells and also organizes internal membranous compartments. In particular, it interacts with membranes in intracellular transport of material in mammalian cells, yeast or plant cells. Tubular membrane intermediates, pulled along microtubule tracks, are involved during these processes, and destabilize into vesicles. While the role of actin in this destabilization process is still debated, literature also provide examples of membranous structures stabilization by actin. To directly address this apparent contradiction, we mimic the geometry of tubular intermediates with preformed membrane tubes. The growth of an actin sleeve at the tube surface is monitored spatio-temporally. Depending on network cohesiveness, actin is able to stabilize, or maintain membrane tubes under pulling. Indeed, on a single tube, thicker portions correlate with the presence of actin. Such structures relax over several minutes, and may provide enough time and curvature geometries for other proteins to act on tube stability.

biophysics↗

bioRxiv2018

A direct proof that sole actin dynamics drive membrane deformations

Cell membrane deformations are crucial for proper cell function. Specialized protein assemblies initiate inward or outward membrane deformations that turn into, for example, filopodia or endocytic intermediates. Actin dynamics and actin-binding proteins are involved in this process, although their detailed role remains controversial. We show here that a dynamic, branched actin network is sufficient, in absence of any membrane-associated proteins, to initiate both inward and outward membrane deformation. With actin polymerization triggered at the membrane of liposomes, we produce inward filopodia-like structures at low tension, while outward endocytosis-like structures are robustly generated regardless of tension. Our results are reminiscent of endocytosis in mammalian cells, where actin polymerization forces are required when membrane tension is increased, and in yeast, where they are always required to overcome the opposing turgor pressure. By combining experimental observations with physical modeling, we propose a mechanism for actin-driven endocytosis under high tension or high pressure conditions.

biophysics↗