| In the natural world,biological systems,as non-equilibrium open systems,constantly interact with their external environment through the exchange of information,matter,and energy,showcasing their inherent characteristics.Cells,the basic units of life,respond to external stimuli by engaging in decision-making processes that are rich in dynamic and thermodynamic properties.However,the internal mechanisms behind these processes remain not fully understood.A major challenge in contemporary biophysics and the science of complex systems is to comprehend how these processes maintain order and function under conditions far from equilibrium.In recent years,non-equilibrium statistical physics has offered significant insights into addressing this challenge.This includes using potential energy landscapes and probabilistic flow frameworks to analyze non-equilibrium networks,as well as employing high-dimensional state spaces to understand macroscopic emergent phenomena.Yet,applying these advanced theories to thoroughly dissect the non-equilibrium principles in life’s processes and to build a unified theoretical framework still poses many difficulties.To advance our understanding of this complex scientific challenge,this study integrates forefront theories of non-equilibrium statistical physics into life sciences research.We have chosen Saccharomyces cerevisiae’s response to pheromone signaling as our research model.Employing sophisticated microfluidic single-cell culture techniques coupled with single-molecule fluorescence imaging,we have acquired data with high temporal resolution,capturing multidimensional dynamic responses.Our quantitative analysis of this dataset revealed a pattern of significant non-equilibrium steady-state phenomena.These phenomena are not mere random occurrences;instead,they epitomize inherent regularities discernible within a comprehensive dataset.These steady-state behaviors,observed under varied stimulus conditions,consistently and reproducibly manifest,uncovering universal principles that transcend the cellular level,thus offering multidimensional insights into the complexity of these biological systems.In delving into the pheromone-induced fate decision-making processes of yeast cells,this study elucidates the multilayered physical mechanisms underlying cellular fate decisions,paving the way for novel multidimensional approaches to understanding non-equilibrium phenomena in biological processes.This thesis offers a quantitative dissection of the fate decision-making pathways in yeast cells subjected to pheromonal cues,parsed into three principal avenues:1.Gene Expression Fate Decision under Non-Equilibrium Steady States:In this section,we synergize biological and physical methodologies to deconstruct the dynamic perturbations of the Fus3 protein,a constituent of the MAPK ensemble,under variegated pheromonal intensities.Commencing with flow cytometry,we pinpointed the pivotal threshold catalyzing cellular responsiveness to pheromonal gradations.This preliminary step was succeeded by an examination of individual yeast cells’multifaceted reactions to disparate pheromone levels,delineating the non-equilibrium steady states manifest during the response trajectory.These states furnished a quantitative portrayal of divergent gene expression fates,encompassing dual Fus3expression states coexisting within the nucleus and cytoplasm.Employing a hidden Markov model to fit fluorescence intensity traces enabled the extraction of bifurcated gene expression modalities and their associated transition probabilities.Concomitantly,we meticulously charted salient dynamics,such as state transition velocities,occupancy durations,and potential energy barriers.Significantly,these parameters under assorted pheromonal concentrations revealed a contrarian relationship between barrier heights and residency periods,shedding light on the mechanisms engendering multiple steady states.Additionally,our foray into the regulatory impact of feedback mechanisms on the gene expression potential landscape has surfaced novel discernments into the determinants of yeast cell destiny.In sum,this research clarifies the modulation of the non-equilibrium potential landscape through pheromonal concentration,which in turn irrevocably sculpts the cellular fate by reconfiguring the signal network’s intrinsic linkages.The revelations extend beyond offering a novel multi-dimensional vantage point within non-equilibrium biophysics;they are pivotal in enhancing our grasp of the intricate dynamics governing cellular fate determination.2.Non-Equilibrium Steady-State Decision-Making in Yeast Polar Growth.In this section,we probed the dynamic aspects of polar growth in yeast cells employing microfluidic single-cell culture apparatus and live microscopic tracking.Cell segmentation was performed using a circular filling methodology,and a quasi-harmonic mean(H_n)served as a quantifier,revealing heterogeneous morphological transformations and four distinct growth rates and morphological fates at varying growth junctures.The morphological system’s temporal evolution during polar growth was quantified by fitting cell shape time-series data with Hidden Markov Models.A novel theoretical construct rooted in non-equilibrium physics was formulated to decode the experimental observations of cellular fates.This construct harnesses non-equilibrium landscape and probabilistic flux theories for a quantitative elucidation of yeast cells’pair response kinetics within a biological milieu.To substantiate the fates pertaining to cellular growth velocities and morphology,a bicolor fluorescent system(CDC24-GFP＿FUS3-RFP)was engineered.This visualization tool permitted direct observation and analytical quantification of the interplay between cell growth rates and morphologies,offering pivotal insights into the molecular mechanisms underpinning cell fate determinations.Our empirical findings furnish the inaugural comprehensive quantitative corroboration of instantaneous concordance among intracellular signaling,physiological proliferation,and morphological functions.This evidence substantiates our postulated molecular schema governing the manifestation of multiple cellular destinies.Moreover,the Gillespie algorithmic model was invoked to emulate the biochemical response of yeast cells to pheromonal induction,affirming the importance and logical coherence of diverse cellular phenotype fates in characterizing the aggregate cellular comportment.3.Study of Non-Equilibrium Dynamics and Thermodynamics in Yeast Cell Fate Decision under Non-Equilibrium Steady States.Employing time-series data from empirical observations alongside Hidden Markov Model,we performed an in-depth quantitative examination of the dynamical shifts within the cellular morphological framework.The ensuing statistical portrayal elucidated the stability landscapes of the attractor states.By quantifying the vorticity flux undermining extant attractors,we unearthed the underpinnings of non-equilibrium dynamics that steer morphological fates from a multiplicity of states toward a singular preeminent one.Our findings indicate that escalated pheromonal doses precipitate pronounced chemical potential disparities,thus intensifying the chemical flux.Such augmentation not only bolsters the system’s net influx but also unsettles previously maintained detailed balance states.Probing the thermodynamic expenditure requisite for the sustenance of cellular morphological constancy shed light on the thermodynamic genesis of the entropy production rate during the phase transitions of non-equilibrium morphological configurations.Further,from a thermodynamic stance,we explored the energy dissipation patterns inherent in cellular decisions under varied stimuli,discerning that heightened stimuli correlate with increased energy depletion.This offers novel insights for the modulation of molecular machinations via potent negative feedback conduits.Additionally,the temporal series irreversibility has been validated as an efficacious predictor for non-equilibrium phase shifts.Culminating in the synergistic affirmation through biological experimentation and physical theoretical frameworks,as well as the interlocking dynamical and thermodynamical analysis,our study corroborates the applicability and precision of non-equilibrium statistical physics in the quantitative depiction of life systems.The bridging of physical insights spanning molecular to cellular dimensions is poised to significantly influence the trajectory of future inquiries into the non-equilibrium characteristics of cell fate decisions within animate biological matrices. |
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