Investigation Of The Regulation Of Cell Behaviors By Macromolecular Dynamics In Living Cells

The translocation of biological macromolecules in living cells plays an important role in maintaining the normal function of cells.However,how the subcellular structure affects the dynamics of macromolecules is unknown.In addition,at the cellular level,cells will undergo crawling,apoptosis and other behaviors.In these processes,the significance of macromolecular dynamics in living cells is unknown.Using single-molecule fluorescence tracking technology,we explored the diffusion of quantum dots,the active transport process of endocytic vesicles,the relationship between intracelluar diffusion and movement behavior during keratocyte migration,and the relationship between chromatin dynamics and nuclear deformationduring apoptosis.There are mainly the following four aspects of progress:Intracellular diffusion is critical for molecule translocation in cytoplasm and mediates many important cellular processes.The real behaviors of 3D diffusion of molecules in cytoplasm are still unclear.Here,we have built a 3D single-particle tracking(SPT)microscopy and studied the 3D diffusionof quantum dots(QDs)in adherent A549 cells.Notably,we found that the intracellular diffusion of QDs is quasi-2D,with the axial motion being severely confned.Further investigations demonstrated that disrupting the cytoskeleton component or endoplasmic reticulum(ER)does not alter the quasi-2D diffusion pattern,although ER reduces the diffusion rates and slightly relieves the constraint in the axial diffusion.The preferred quasi-2D diffusion is quite robust and attributed to the complex cytoarchitectures in the flat adherent cells.With the aid of 3D SPT method,the quasi-2D diffusion in cells was revealed,shedding new light on the physical nature of cytoplasm.Vesicle transport in cells is crucial for biomolecule translocations,which is regulated by both complex cytoarchitectures and active driving forces.The real 3D characteristics of vesicle transport in cells is still unclear,especially the temporal-spatial dynamics and its regulatory mechanism.Here,with 3D SPT,we comprehensively studied the physical properties of the intracellular transport dynamics of endocytic vesicles.Temporally,we observed that 3D intracellular transport shows a thermal-dominated isotropic constrained motion at a timescale shorter than 0.1 s,which turns to an active-dominated quasi-2D motion over 0.1 s.Spatially,the lateral motion shows to be heterogeneous with the peripheral region being faster than the perinuclear region,while the axial motion is homogeneous across the cell.We further confirmed that the microtubule-based directed motion leads to the long-time anisotropy and the lateral heterogeneity of the vesicle transport,whereas the actin filaments impose an isotropic constrain on the vesicles.Strikingly,inside the vesicles,we observed that the endocytic fluorescent particles may make diffusing motions on their inner membranes when microtubules are absent in cells,suggesting that the endocytic cargos are normally localized at the inner membranes of vesicles through a physical connection to the microtubules outside during active intracellular transport.Cell migration provides important functions in multiple biological processes,requiring the orchestration of numerous intracellular molecules and subcellular structures in time and space.However,how cells coordinate these complex factors to effectively control the migrative behaviours is unknown.Here,by exploring the intracellular diffusion and the coupled 3D lamellipodium structure,we uncovered a reversibly symmetry-breaking mechanism in which two distinct migration modes are exploited by cells to modulate the speeds.Compared with normally moving cells with the flat lamellipodia and rapid intracellular diffusion,cells at reduced speeds are found to swell at front lamellipodia but be compressed at the rear region,in which macromolecules are squeezed out from the rear and concentrated in the front lamellipodia,reducing the diffusion rates and generating a discontinuous diffusion map.As such,the demixing phase separation of the macromolecules like the actin subunits is proposed to result in local recycling of actin subunits in the front lamellipodia,which could slow down the overall actin protrusion rates and cell migration.We further demonstrated that this migration mode is reversable to the normally moving mode when the cell speeds increase.Moreover,an analogous left-right symmetry-breaking in both intracellular diffusion and lamellipodium structure is found in the turning cells.These finding reveals a new regulation mechanism of spatiotemporal coordination between intracellular dynamics and lamellipodium structures in cell migrations.The process of chromatin replication and transcription in the nucleus is accompanied by changes in the chromatin structures.The morphology of the cell nucleus may also change during the relevant processes.During the change of chromatin state,how chromatin dynamics changes and how this affects nuclear deformation is unknown.Here,we explored the chromatin dynamics and nuclear deformation processes during apoptosis.By labeling the telomere and H2 B in the nucleus,we tracked the movement of telomeres and monitored the morphological changes of the nucleus at the same time.We found that chromatin dynamics is accelerated during drug-induced apoptosis,which triggers nuclear invagination.Furthermore,through time series analysis,it was found that the chromatin dynamics is accelerated earlier than the morphological changes of the nucleus.Subsequently,the caspase inhibitor z VAD was used in combination with Cis(cisplatin)and CPT(Camptothecin),respectively,and it was found that DNA breakage caused by CPT would accelerate the chromatin dynamics and trigger the nuclear invagination.In addition,we found that the acceleration of chromatin dynamics is caused by the change of chromatin state,rather than the destruction of Lamin A.Finally,increasing the ratio of heterochromatin can inhibit the acceleration of chromatin dynamics and the generation of nuclear invagination.Regulating the chromatin state can change the dynamics of the nucleus and protect the nucleus morphology.This provides new ideas for inhibiting the process of apoptosis or even tumor metastasis.