Cytoplasmic streaming | EPFL Graph Search

Cytoplasmic streaming, also called protoplasmic streaming and cyclosis, is the flow of the cytoplasm inside the cell, driven by forces from the cytoskeleton. It is likely that its function is, at least in part, to speed up the transport of molecules and organelles around the cell. It is usually observed in large plant and animal cells, greater than approximately 0.1 mm. In smaller cells, the diffusion of molecules is more rapid, but diffusion slows as the size of the cell increases, so larger cells may need cytoplasmic streaming for efficient function. The green alga genus Chara possesses some very large cells, up to 10 cm in length, and cytoplasmic streaming has been studied in these large cells. Cytoplasmic streaming is strongly dependent upon intracellular pH and temperature. It has been observed that the effect of temperature on cytoplasmic streaming created linear variance and dependence at different high temperatures in comparison to lo

Circadian dynamics of RNA localisation in the mammalian liver

Clémence Yumie Syloun Hurni

Gene expression in eukaryotes is a complex multi-step process. It starts in the nucleus with transcription, the synthesis of a mRNA copy from a DNA template. While in the nucleus, the RNA transcript is subject to multiple co- and post-transcriptional modifications, including splicing, capping, polyadenylation, and assembly into ribonucleoprotein complexes. Correctly processed mRNA is exported to the cytoplasm and translated by ribosomes to form a chain of amino acids: the protein. The mRNA lifetime in the cytoplasm is determined by the activity of a distinct set of RNA Binding Proteins, enzymes, and functional RNAs, which promote stability or degradation. Each step of the RNA life cycle is tightly regulated to ensure proper cellular function. The kinetic rates governing these steps are dynamic and notably adapt to the daily fluctuations of the environment related to the alternance of day and night. Indeed, most organisms possess an internal timing system called the circadian clock. The clock is a genetically encoded self-sustained transcriptional-translational feedback loop, which operates in almost every cell and tissue of the body. It controls the temporal gene expression program to synchronise cellular and physiological functions to the external world. In this thesis, I explore the transcriptome of the mouse liver by combining RNA-sequencing, mathematical modelling, and single-molecule RNA-FISH (smFISH), with an emphasis on the spatio-temporal organisation of RNA expression at the subcellular and tissue scales. I first investigate how RNA are differentially localised at the scale of the liver tissue. Hepatocytes are arranged in structural and functional units called lobules, and carry out different physiological functions depending on their spatial position within the lobule. In this collaborative work, we characterised spatio-temporal gene expression profiles, and showed that that while the expression of hundreds of genes is dually orchestrated by time and space, the circadian core clock is expressed uniformly within the liver lobule, and is therefore robust to the heterogeneous microenvironment. Second, I explore the RNA localisation at the scale of a hepatocyte. The subcellular distributions of RNA in different compartments (here, the nucleus and the cytoplasm), are dictated by the balance of a synthesis term and a decay term. To quantify the kinetic parameters driving nuclear and cytoplasmic mRNA accumulation, I sequenced RNA from both cellular fractions from mouse livers sampled at different times of the day. Using a mathematical model describing rhythmic pre-mRNA and mRNA profiles, I could estimate the nuclear export rates and cytoplasmic degradation rates of

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1400 genes. Nuclear export occurs on a much shorter time-scale than cytoplasmic degradation, and nuclear lifetime has only a minor contribution to the total RNA lifetime. However, a subset of metabolic genes remain in the nucleus for more than one hour (up to four hours), which accounts for the long phase delay between the peak times of transcription and of cytoplasmic accumulation. Furthermore, nuclear export contributes to the modulation and generation of rhythmic profiles of ~10% of the cycling nuclear mRNA. This study provides a comprehensive estimation of the nuclear and cytoplasmic life times in the liver and contributes to a better understanding of the dynamic regulation of the transcriptome during the feeding-fasting cycle.

EPFL2021

Molecular Modeling of Membrane Embedded Proteins

Thomas Lemmin

Over the past years, molecular modeling and simulation techniques have had a major impact on experimental life sciences. They are capable of providing accurate insight into microscopic mechanisms, which are usually difficult to investigate experimentally. Moreover, the integration of experimental data with molecular modeling appears to be a promising strategy to better understand complex biological processes. In this thesis, molecular dynamics simulation has been used in combination with experimental data to investigate two transmembrane proteins: (i) the bacterial chemoreceptor PhoQ and (ii) the Amyloid Precursor Protein (APP). (i) Bacterial two-component system PhoQ and bacterial membranes. Two-component systems (TCSs) are signaling complexes essential for bacterial survival and virulence. PhoQ is the histidine kinase chemoreceptor of the PhoQ-PhoP tandem machine that detects the concentration of cationic species at the inner membrane of Gram-negative bacteria. A full understanding of the PhoQ signal transduction mechanism is currently hindered by the lack of a complete atomistic structure. In this thesis project, the first structural model of the transmembrane (TM) portion of PhoQ from Escherichia coli was assembled, by using molecular simulations integrated with cross-linking disulfide scanning data. Its structural and dynamic features induce a concerted displacement of the TM helices at the periplasmic side, which modulates a rotation at the cytoplasmic end. This supports the idea that signal transduction is promoted through a combination of scissoring and rotational movements of the TM helices. Knowledge of this complex mechanism is essential in order to understand how the chemical stimuli sensed by the periplasmic sensor domain trigger, via the relay of the HAMP domain, the histidine auto-phosphorylation and kinase/phosphatase activity at the cytoplasmic end. The PhoQ sensor domain lies in close proximity to the membrane. Interaction with anionic lipids, such as phosphophatidylglycerols (PG) and cardiolipins (CL), are thought to play a key role in PhoQ activity. Present in bacterial and mitochondrial membranes, cardiolipins have a unique dimeric structure, which carries up to two charges, i.e. one per phosphate group, and under physiological conditions, can be unprotonated or singly protonated. Exhaustive models and characterization of cardiolipins are to-date scarce; therefore an ab initio parameterization of cardiolipin species for molecular simulation consistent with commonly used force fields is proposed here. Molecular dynamics (MD) simulations based on these models indicate a protonation-dependent lipid packing. A noteworthy interaction with solvating mono- and divalent cations is also observed. The proposed models will contribute to the biophysical and biochemical characterizations of bacterial and mitochondrial membranes and membrane-embedded proteins. (ii) Structural and dynamic properties of the Amyloid Precursor Protein. The Amyloid Precursor Protein (APP) is a type I membrane glycoprotein present at the neuronal synapsis. The proteolytic cleavage of its C-terminal segment produces amyloid-β (Aβ) peptides of different lengths, the deposition of which is an early indicator of Alzheimer"s disease (AD). Recently, the backbone structure of the APP transmembrane (TM) domain in detergent micelles was determined by nuclear magnetic resonance (NMR, independently by two different experimental groups). The TM conformations of these two structures are however markedly different. One is characterized by a highly kinked α-helix, whereas the other is mainly straight. Molecular dynamics simulations showed that the APP TM region is highly flexible and its secondary structure is influenced by the surrounding lipid environment. The size of the embedding detergent micelles strongly affects the conformation of the APP α-helix, with solvation being the main driving force for the development of a helical curvature. Once embedded in a membrane bilayer, APP systematically prefers a straight helical conformation. This is also confirmed when analyzing in silico the atomistic APP population observed in double electron-electron resonance (DEER) spectroscopy. In summary, the APP transmembrane domain is highly flexible due to the presence of glycine residues and can readily respond to the lipid environment, a property that might be critical for proteolytic processing by γ-secretase enzymes. The presented thesis work clearly shows how molecular simulations and their interplay with available experimental input can help advance the understanding of the mechanism of complex biological systems and processes on a molecular scale. These results, in particular, go well beyond the current understanding of the functioning of two transmembrane proteins relevant for human health. Furthermore, the computational approaches and procedures developed in these projects will hopefully promote novel integrated strategies for investigating biological systems.

EPFL2013

Assembly of the Transmembrane Domain of E. coli PhoQ Histidine Kinase: Implications for Signal Transduction from Molecular Simulations

Matteo Dal Peraro, Thomas Lemmin

The PhoQP two-component system is a signaling complex essential for bacterial virulence and cationic antimicrobial peptide resistance. PhoQ is the histidine kinase chemoreceptor of this tandem machine and assembles in a homodimer conformation spanning the bacterial inner membrane. Currently, a full understanding of the PhoQ signal transduction is hindered by the lack of a complete atomistic structure. In this study, an atomistic model of the key transmembrane (TM) domain is assembled by using molecular simulations, guided by experimental cross-linking data. The formation of a polar pocket involving Asn202 in the lumen of the tetrameric TM bundle is crucial for the assembly and solvation of the domain. Moreover, a concerted displacement of the TM helices at the periplasmic side is found to modulate a rotation at the cytoplasmic end, supporting the transduction of the chemical signal through a combination of scissoring and rotational movement of the TM helices.

Public Library Science2013