Cytoplasmic concentration: an old question and a new parameter in cell biology

doi:10.12211/2096-8280.2024-086

Abstract

Abstract:

Cytoplasmic concentration is an important parameter in cell physiology, influencing almost all biochemical reactions, playing key roles in regulating various cellular biological processes. However, studying cytoplasmic concentration has been particularly challenging due to its inherent complexity, the difficulty of direct manipulation, and the lack of precise measurement techniques in intact cells. In recent years, advances in microscopy, microfluidics, and synthetic biology have led to the development of novel tools for studying cytoplasmic concentration, such as Quantitative Phase Microscopy, Stimulated Raman Scattering Microscopy, and Genetically Encoded Multimers for single-particle tracking, etc. These tools have enabled researchers to explore the regulation of cytoplasmic concentration, the mechanism of concentration homeostasis, and their influences on cellular physiology, providing deeper insights into their roles in physiological regulations. In this review, we explore both historical and recent advances in methods and overview data regarding cytoplasmic concentration, summarize the molecular and systematic mechanisms that govern its homeostatic regulation, and highlight its roles in physiological and biochemical processes. Specifically, we discuss the key biological processes that influence cytoplasmic concentration, including mitotic swelling, genome dilution, protein synthesis and degradation, and importantly, the heterogeneity of cytoplasmic concentration that arises from local subcellular structure and thermodynamic fluctuation. Furthermore, we expand on the connections between cytoplasmic concentration, cellular aging, signal transduction, cellular differentiation, and microtubule assembly dynamics. Additionally, we explore the theoretical interpretation of cytoplasmic concentration in reaction kinetics and homeostatic regulation, providing evidence from both experimental and theoretical studies on the prevalence of diffusion-limited reactions in biological systems. Despite these advances, significant challenges remain in fully understanding the underlying mechanisms of cytoplasmic concentration homeostasis, its complex interactions with other physiological processes, and its potential applications in synthetic biology. Research on cytoplasmic concentration is rapidly evolving into an active field of study, promising major breakthroughs in the understandings of the fundamentals of cellular life, improvement of human health, and engineering of synthetic cells.

Key words: cytoplasmic concentration, homeostatic control, protein synthesis, biomass, reaction rate, negative feedback regulation, diffusion limit

摘要：

细胞质浓度是细胞生理的重要参数，影响几乎所有生化反应，参与调节细胞生物学过程。近年来，随着显微技术、微流控技术以及合成生物学的发展，研究细胞质浓度的工具不断涌现，促进了对细胞质浓度稳态的调控机理及细胞质生物学的探索，增强了对细胞质浓度参与细胞生理调控的理解。本文介绍了监测细胞质浓度的新方法、新数据，归纳了细胞质浓度稳态调控机制，总结了细胞质浓度在生理生化过程中发挥的作用，从理论和实验角度探讨了细胞质浓度异质性的功能和调控机制，介绍并扩充了细胞质浓度在反应速率、稳态调节中的理论研究。目前对于细胞质浓度的研究在稳态决定机制、与其他生理过程的相互作用及合成生物学中的应用方面还有诸多难题亟待突破。细胞质浓度的探究正逐渐形成一个活跃的研究领域，在多学科交叉、理解生命、人类健康、合成细胞等方面将有重大进展。

关键词: 细胞质浓度, 稳态调控, 蛋白质合成, 生物质, 反应速率, 负反馈调节, 扩散受限

CLC Number:

Q816

Qian LI, E. Ferrell James, Yuping CHEN. Cytoplasmic concentration: an old question and a new parameter in cell biology[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2024-086.

李倩, James E. Ferrell, 陈于平. 细胞质浓度：细胞生物学的老问题、新参数[J]. 合成生物学, DOI: 10.12211/2096-8280.2024-086.

Figures/Tables 4

References 117

1	WILSON E B. The Cell in Development and Inheritance [M]. Macmillan, 1896.
2	MILO R, PHILLIPS R. Cell biology by the numbers [M]. Garland Science, 2015.
3	NATHANS D, SMITH H O. Restriction endonucleases in the analysis and restructuring of dna molecules [J]. Annu Rev Biochem, 1975, 44(Volume 44, 1975): 273-93.
4	CHIEN A, EDGAR D B, TRELA J M. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus [J]. Journal of bacteriology, 1976, 127(3): 1550-7.
5	ALBERTS B, JOHNSON A, LEWIS J, et al. Molecular Biology of the Cell [M]. Garland Science, 2014.
6	JACKSON D A, SYMONS R H, BERG P. Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli [J]. Proc Natl Acad Sci U S A, 1972, 69(10): 2904-9.
7	PRASHER D C, ECKENRODE V K, WARD W W, et al. Primary structure of the Aequorea victoria green-fluorescent protein [J]. Gene, 1992, 111(2): 229-33.
8	JINEK M, CHYLINSKI K, FONFARA I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity [J]. Science, 2012, 337(6096): 816-21.
9	ELOWITZ M B, LEIBLER S. A synthetic oscillatory network of transcriptional regulators [J]. Nature, 2000, 403(6767): 335-8.
10	TRUNNELL N B, POON A C, KIM S Y, et al. Ultrasensitivity in the Regulation of Cdc25C by Cdk1 [J]. Mol Cell, 2011, 41(3): 263-74.
11	PARDEE A B, JACOB F, MONOD J. The genetic control and cytoplasmic expression of "Inducibility" in the synthesis of β-galactosidase by E. coli [J]. Journal of Molecular Biology, 1959, 1(2): 165-78.
12	KANEHISA M. Enzyme Annotation and Metabolic Reconstruction Using KEGG [J]. Methods Mol Biol, 2017, 1611: 135-45.
13	CHEN Y, HUANG J H, PHONG C, et al. Viscosity-dependent control of protein synthesis and degradation [J]. Nat Commun, 2024, 15(1): 2149.
14	ALRIC B, FORMOSA-DAGUE C, DAGUE E, et al. Macromolecular crowding limits growth under pressure [J]. Nat Phys, 2022, 18(4): 411-6.
15	MOLINES A T, LEMIERE J, GAZZOLA M, et al. Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization [J]. Dev Cell, 2022, 57(4): 466-79 e6.
16	WEBB B A, CHIMENTI M, JACOBSON M P, et al. Dysregulated pH: a perfect storm for cancer progression [J]. Nat Rev Cancer, 2011, 11(9): 671-7.
17	ELOWITZ M B, SURETTE M G, WOLF P E, et al. Protein mobility in the cytoplasm of Escherichia coli [J]. Journal of bacteriology, 1999, 181(1): 197-203.
18	BEGASSE M L, LEAVER M, VAZQUEZ F, et al. Temperature Dependence of Cell Division Timing Accounts for a Shift in the Thermal Limits of C. elegans and C. briggsae [J]. Cell Rep, 2015, 10(5): 647-53.
19	PERSSON L B, AMBATI V S, BRANDMAN O. Cellular Control of Viscosity Counters Changes in Temperature and Energy Availability [J]. Cell, 2020, 183(6): 1572-85 e16.
20	MINTON A P. Implications of macromolecular crowding for protein assembly [J]. Curr Opin Struct Biol, 2000, 10(1): 34-9.
21	HARRIS R F. Effect of Water Potential on Microbial Growth and Activity [M]. Water Potential Relations in Soil Microbiology. 2015: 23-95.
22	CHAKRABARTI B, RACHH M, SHVARTSMAN S Y, et al. Cytoplasmic stirring by active carpets [J]. Proc Natl Acad Sci U S A, 2024, 121(30): e2405114121.
23	BRANGWYNNE C P, KOENDERINK G H, MACKINTOSH F C, et al. Cytoplasmic diffusion: molecular motors mix it up [J]. The Journal of cell biology, 2008, 183(4): 583-7.
24	BRANGWYNNE C P, KOENDERINK G H, MACKINTOSH F C, et al. Intracellular transport by active diffusion [J]. Trends in cell biology, 2009, 19(9): 423-7.
25	NEUROHR G E, AMON A. Relevance and Regulation of Cell Density [J]. Trends in cell biology, 2020, 30(3): 213-25.
26	DILL K A, GHOSH K, SCHMIT J D. Physical limits of cells and proteomes [J]. Proc Natl Acad Sci U S A, 2011, 108(44): 17876-82.
27	ELLIS R J. Macromolecular crowding: obvious but underappreciated [J]. Trends Biochem Sci, 2001, 26(10): 597-604.
28	GUO M, PEGORARO A F, MAO A, et al. Cell volume change through water efflux impacts cell stiffness and stem cell fate [J]. Proc Natl Acad Sci U S A, 2017, 114(41): E8618-E27.
29	CADART C, BARTZ J, OAKS G, et al. Polyploidy in Xenopus lowers metabolic rate by decreasing total cell surface area [J]. Current biology : CB, 2023, 33(9): 1744-52 e7.
30	CADART C, VENKOVA L, RECHO P, et al. The physics of cell-size regulation across timescales [J]. Nature Physics, 2019, 15(10): 993-1004.
31	MINTON A P. The influence of macromolecular crowding and macromolecular confinement on biochemical reactions in physiological media [J]. The Journal of biological chemistry, 2001, 276(14): 10577-80.
32	LEMIERE J, REAL-CALDERON P, HOLT L J, et al. Control of nuclear size by osmotic forces in Schizosaccharomyces pombe [J]. Elife, 2022, 11: e76075.
33	HARTWELL L H. Periodic density fluctuation during the yeast cell cycle and the selection of synchronous cultures [J]. Journal of bacteriology, 1970, 104(3): 1280-5.
34	RAHMAN Y E, ELSON D L, CERNY E A. Studies on the mechanism of erythrocyte aging and destruction. I. Separation of rat erythrocytes according to age by Ficoll gradient centrifugation [J]. Mech Ageing Dev, 1973, 2(2): 141-50.
35	SEGAL A W, FORTUNATO A, HERD T. A rapid single centrifugation step method for the separation of erythrocytes, granulocytes and mononuclear cells on continuous density gradients of Percoll [J]. J Immunol Methods, 1980, 32(3): 209-14.
36	CATSIMPOOLAS N. Methods of cell separation [M]. Springer Science & Business Media, 2012.
37	KRUGER N J. The Bradford method for protein quantitation [J]. The protein protocols handbook, 2009: 17-24.
38	NOBLE J E, BAILEY M J A. Chapter 8 Quantitation of Protein [M]//BURGESS R R, DEUTSCHER M P. Methods in Enzymology. Academic Press. 2009: 73-95.
39	PETERSON M S, PATKAR A Y, SEO J H. Flow cytometric analysis of total protein content and size distributions of recombinant Saccharomyces cerevisiae [J]. Biotechnology Techniques, 1992, 6(3): 203-6.
40	CRISSMAN H A, STEINKAMP J A. Rapid, simultaneous measurement of DNA, protein, and cell volume in single cells from large mammalian cell populations [J]. The Journal of cell biology, 1973, 59(3): 766-71.
41	ROTH B L, POOT M, YUE S T, et al. Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain [J]. Appl Environ Microbiol, 1997, 63(6): 2421-31.
42	NASH R, TOKIWA G, ANAND S, et al. The WHI1+ gene of Saccharomyces cerevisiae tethers cell division to cell size and is a cyclin homolog [J]. EMBO J, 1988, 7(13): 4335-46.
43	ROSEBROCK A P. Analysis of the Budding Yeast Cell Cycle by Flow Cytometry [J]. Cold Spring Harb Protoc, 2017, 2017(1): pdb.prot088740.
44	BLATTER L A. [16] Cell volume measurements by fluorescence confocal microscopy: Theoretical and practical aspects [M]. Methods in Enzymology. Academic Press. 1999: 274-95.
45	XIE S, SKOTHEIM J M. A G1 Sizer Coordinates Growth and Division in the Mouse Epidermis [J]. Current biology : CB, 2020, 30(5): 916-24 e2.
46	ZLOTEK-ZLOTKIEWICZ E, MONNIER S, CAPPELLO G, et al. Optical volume and mass measurements show that mammalian cells swell during mitosis [J]. Journal of Cell Biology, 2015, 211(4): 765-74.
47	GROVER W H, BRYAN A K, DIEZ-SILVA M, et al. Measuring single-cell density [J]. Proc Natl Acad Sci U S A, 2011, 108(27): 10992-6.
48	SON S, KANG J H, OH S, et al. Resonant microchannel volume and mass measurements show that suspended cells swell during mitosis [J]. Journal of Cell Biology, 2015, 211(4): 757-63.
49	ICHA J, WEBER M, WATERS J C, et al. Phototoxicity in live fluorescence microscopy, and how to avoid it [J]. BioEssays, 2017, 39(8): 1700003.
50	DEMCHENKO A P. Photobleaching of organic fluorophores: quantitative characterization, mechanisms, protection [J]. Methods Appl Fluoresc, 2020, 8(2): 022001.
51	PARK Y, DEPEURSINGE C, POPESCU G. Quantitative phase imaging in biomedicine [J]. Nature Photonics, 2018, 12(10): 578-89.
52	CURL C L, BELLAIR C J, HARRIS T, et al. Refractive index measurement in viable cells using quantitative phase-amplitude microscopy and confocal microscopy [J]. Cytometry A, 2005, 65(1): 88-92.
53	BON P, MAUCORT G, WATTELLIER B, et al. Quadriwave lateral shearing interferometry for quantitative phase microscopy of living cells [J]. Opt Express, 2009, 17(15): 13080-94.
54	CREATH K, GOLDSTEIN G. Dynamic quantitative phase imaging for biological objects using a pixelated phase mask [J]. Biomed Opt Express, 2012, 3(11): 2866-80.
55	GARDINER D J. Introduction to Raman scattering [M]. Practical Raman Spectroscopy. Springer. 1989: 1-12.
56	FREUDIGER C W, MIN W, SAAR B G, et al. Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy [J]. Science, 2008, 322(5909): 1857-61.
57	CHENG J X, XIE X S. Vibrational spectroscopic imaging of living systems: An emerging platform for biology and medicine [J]. Science, 2015, 350(6264): aaa8870.
58	HILL A H, MANIFOLD B, FU D. Tissue imaging depth limit of stimulated Raman scattering microscopy [J]. Biomed Opt Express, 2020, 11(2): 762-74.
59	FU D, LU F K, ZHANG X, et al. Quantitative chemical imaging with multiplex stimulated Raman scattering microscopy [J]. J Am Chem Soc, 2012, 134(8): 3623-6.
60	OH S, LEE C, YANG W, et al. Protein and lipid mass concentration measurement in tissues by stimulated Raman scattering microscopy [J]. Proc Natl Acad Sci U S A, 2022, 119(17): e2117938119.
61	GARNER R M, MOLINES A T, THERIOT J A, et al. Vast heterogeneity in cytoplasmic diffusion rates revealed by nanorheology and Doppelganger simulations [J]. Biophys J, 2023, 122(5): 767-83.
62	HUANG W Y C, CHENG X, FERRELL J E, JR. Cytoplasmic organization promotes protein diffusion in Xenopus extracts [J]. Nat Commun, 2022, 13(1): 5599.
63	PHILLIPS R, KONDEV J, THERIOT J, et al. Physical biology of the cell [M]. Garland Science, 2012.
64	DELARUE M, BRITTINGHAM G P, PFEFFER S, et al. mTORC1 Controls Phase Separation and the Biophysical Properties of the Cytoplasm by Tuning Crowding [J]. Cell, 2018, 174(2): 338-49 e20.
65	SHU T, MITRA G, ALBERTS J, et al. Mesoscale molecular assembly is favored by the active, crowded cytoplasm [J]. bioRxiv, 2023.
66	KRICHEVSKY O, BONNET G. Fluorescence correlation spectroscopy: the technique and its applications [J]. Reports on Progress in Physics, 2002, 65(2): 251-97.
67	HUANG W Y C, BOXER S G, FERRELL J E, JR. Membrane localization accelerates association under conditions relevant to cellular signaling [J]. Proc Natl Acad Sci U S A, 2024, 121(10): e2319491121.
68	AXELROD D, KOPPEL D E, SCHLESSINGER J, et al. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics [J]. Biophys J, 1976, 16(9): 1055-69.
69	ZHOU H X, RIVAS G, MINTON A P. Macromolecular crowding and confinement: biochemical, biophysical, and potential physiological consequences [J]. Annual review of biophysics, 2008, 37(1): 375-97.
70	WEISS M, ELSNER M, KARTBERG F, et al. Anomalous subdiffusion is a measure for cytoplasmic crowding in living cells [J]. Biophys J, 2004, 87(5): 3518-24.
71	RIVAS G, MINTON A P. Macromolecular Crowding In Vitro, In Vivo, and In Between [J]. Trends Biochem Sci, 2016, 41(11): 970-81.
72	ALGAR W R, HILDEBRANDT N, VOGEL S S, et al. FRET as a biomolecular research tool - understanding its potential while avoiding pitfalls [J]. Nat Methods, 2019, 16(9): 815-29.
73	BOERSMA A J, ZUHORN I S, POOLMAN B. A sensor for quantification of macromolecular crowding in living cells [J]. Nat Methods, 2015, 12(3): 227-9, 1 p following 9.
74	LIU X, OH S, KIRSCHNER M W. The uniformity and stability of cellular mass density in mammalian cell culture [J]. Front Cell Dev Biol, 2022, 10: 1017499.
75	MIETTINEN T P, KANG J H, YANG L F, et al. Mammalian cell growth dynamics in mitosis [J]. Elife, 2019, 8: e44700.
76	ZHURINSKY J, LEONHARD K, WATT S, et al. A coordinated global control over cellular transcription [J]. Current biology: CB, 2010, 20(22): 2010-5.
77	SWAFFER M P, MARINOV G K, ZHENG H, et al. RNA polymerase II dynamics and mRNA stability feedback scale mRNA amounts with cell size [J]. Cell, 2023, 186(24): 5254-68 e26.
78	NEUROHR G E, TERRY R L, LENGEFELD J, et al. Excessive Cell Growth Causes Cytoplasm Dilution And Contributes to Senescence [J]. Cell, 2019, 176(5): 1083-97 e18.
79	CHEN Y, ZHAO G, ZAHUMENSKY J, et al. Differential Scaling of Gene Expression with Cell Size May Explain Size Control in Budding Yeast [J]. Mol Cell, 2020, 78(2): 359-70 e6.
80	LANZ M C, ZATULOVSKIY E, SWAFFER M P, et al. Increasing cell size remodels the proteome and promotes senescence [J]. Mol Cell, 2022, 82(17): 3255-69 e8.
81	BRANGWYNNE C P, ECKMANN C R, COURSON D S, et al. Germline P granules are liquid droplets that localize by controlled dissolution/condensation [J]. Science, 2009, 324(5935): 1729-32.
82	MOLLIEX A, TEMIROV J, LEE J, et al. Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization [J]. Cell, 2015, 163(1): 123-33.
83	LUO Y, NA Z, SLAVOFF S A. P-Bodies: Composition, Properties, and Functions [J]. Biochemistry, 2018, 57(17): 2424-31.
84	LAFONTAINE D L J, RIBACK J A, BASCETIN R, et al. The nucleolus as a multiphase liquid condensate [J]. Nat Rev Mol Cell Biol, 2021, 22(3): 165-82.
85	HE J, HUO X, PEI G, et al. Dual-role transcription factors stabilize intermediate expression levels [J]. Cell, 2024, 187(11): 2746-66 e25.
86	CHEN Y, FERRELL J E, JR. C. elegans colony formation as a condensation phenomenon [J]. Nat Commun, 2021, 12(1): 4947.
87	ALBERTI S, HYMAN A A. Biomolecular condensates at the nexus of cellular stress, protein aggregation disease and ageing [J]. Nat Rev Mol Cell Biol, 2021, 22(3): 196-213.
88	MINTON A P. The effect of volume occupancy upon the thermodynamic activity of proteins: some biochemical consequences [J]. Mol Cell Biochem, 1983, 55(2): 119-40.
89	KLOSIN A, OLTSCH F, HARMON T, et al. Phase separation provides a mechanism to reduce noise in cells [J]. Science, 2020, 367(6476): 464-8.
90	LUBY-PHELPS K. Cytoarchitecture and Physical Properties of Cytoplasm: Volume, Viscosity, Diffusion, Intracellular Surface Area [M]//WALTER H, BROOKS D E, SRERE P A. Microcompartmentation and Phase Separation in Cytoplasm. Academic Press. 1999: 189-221.
91	MINTON A P. Influence of excluded volume upon macromolecular structure and associations in 'crowded' media [J]. Curr Opin Biotechnol, 1997, 8(1): 65-9.
92	HU Z, CHEN K, XIA Z, et al. Nucleosome loss leads to global transcriptional up-regulation and genomic instability during yeast aging [J]. Genes Dev, 2014, 28(4): 396-408.
93	NEUROHR G E, TERRY R L, SANDIKCI A, et al. Deregulation of the G1/S-phase transition is the proximal cause of mortality in old yeast mother cells [J]. Genes Dev, 2018, 32(15-16): 1075-84.
94	CHEN Y, FUTCHER B. Scaling gene expression for cell size control and senescence in Saccharomyces cerevisiae [J]. Curr Genet, 2021, 67(1): 41-7.
95	MANOHAR S, ESTRADA M E, ULIANA F, et al. Genome homeostasis defects drive enlarged cells into senescence [J]. Mol Cell, 2023, 83(22): 4032-46 e6.
96	AOKI K, TAKAHASHI K, KAIZU K, et al. A quantitative model of ERK MAP kinase phosphorylation in crowded media [J]. Sci Rep, 2013, 3(1): 1541.
97	HUANG J H, CHEN Y, HUANG W Y C, et al. Robust trigger wave speed in Xenopus cytoplasmic extracts [J]. Nat Commun, 2024, 15(1): 5782.
98	JIANG H, SONG C, CHEN C C, et al. Quantitative 3D imaging of whole, unstained cells by using X-ray diffraction microscopy [J]. Proc Natl Acad Sci U S A, 2010, 107(25): 11234-9.
99	PLANTE S, MOON K M, LEMIEUX P, et al. Breaking spore dormancy in budding yeast transforms the cytoplasm and the solubility of the proteome [J]. PLoS Biol, 2023, 21(4): e3002042.
100	SHARMA R, AGARWAL A. Spermatogenesis: An Overview [M]//ZINI A, AGARWAL A. Sperm Chromatin. New York, NY; Springer New York. 2011: 19-44.
101	MAURER F, JOHN T, MAKHRO A, et al. Continuous Percoll Gradient Centrifugation of Erythrocytes-Explanation of Cellular Bands and Compromised Age Separation [J]. Cells, 2022, 11(8).
102	COOPER K L, OH S, SUNG Y, et al. Multiple phases of chondrocyte enlargement underlie differences in skeletal proportions [J]. Nature, 2013, 495(7441): 375-8.
103	BROUHARD G J, RICE L M. Microtubule dynamics: an interplay of biochemistry and mechanics [J]. Nat Rev Mol Cell Biol, 2018, 19(7): 451-63.
104	MIESCH J, WIMBISH R T, VELLUZ M C, et al. Phase separation of +TIP networks regulates microtubule dynamics [J]. Proc Natl Acad Sci U S A, 2023, 120(35): e2301457120.
105	FULTON A B. How crowded is the cytoplasm? [J]. Cell, 1982, 30(2): 345-7.
106	CAMERON I L, KANAL K M, KEENER C R, et al. A mechanistic view of the non-ideal osmotic and motional behavior of intracellular water [J]. Cell Biol Int, 1997, 21(2): 99-113.
107	BAR-EVEN A, NOOR E, SAVIR Y, et al. The moderately efficient enzyme: evolutionary and physicochemical trends shaping enzyme parameters [J]. Biochemistry, 2011, 50(21): 4402-10.
108	MAVROVOUNIOTIS M L, STEPHANOPOULOS G, STEPHANOPOULOS G. Estimation of Upper Bounds for the Rates of Enzymatic Reactions [J]. Chemical Engineering Communications, 2007, 93(1): 211-36.
109	STEITZ T A, SHOHAM M, BENNETT W S. Structural Dynamics of Yeast Hexokinase During Catalysis [J]. Philosophical Transactions of the Royal Society of London Series B, Biological Sciences, 1981, 293(1063): 43-52.
110	KOSHLAND JR D E. The key–lock theory and the induced fit theory [J]. Angewandte Chemie International Edition in English, 1995, 33(23‐24): 2375-8.
111	HOPFIELD J J. Kinetic proofreading: a new mechanism for reducing errors in biosynthetic processes requiring high specificity [J]. Proc Natl Acad Sci U S A, 1974, 71(10): 4135-9.
112	CHOI A A, XU K. Single-Molecule Diffusivity Quantification Unveils Ubiquitous Net Charge-Driven Protein–Protein Interaction [J]. Journal of the American Chemical Society, 2024, 146(15): 10973-8.
113	CHOI A A, ZHOU C Y, TABO A, et al. Single-molecule diffusivity quantification in Xenopus egg extracts elucidates physicochemical properties of the cytoplasm [J]. Proceedings of the National Academy of Sciences, 2024, 121(50): e2411402121.
114	AN S, KUMAR R, SHEETS E D, et al. Reversible compartmentalization of de novo purine biosynthetic complexes in living cells [J]. Science, 2008, 320(5872): 103-6.
115	CHAUDHURI P K, LOW B C, LIM C T. Mechanobiology of Tumor Growth [J]. Chem Rev, 2018, 118(14): 6499-515.
116	ALIBERT C, GOUD B, MANNEVILLE J B. Are cancer cells really softer than normal cells? [J]. Biol Cell, 2017, 109(5): 167-89.
117	DESSARD M, MANNEVILLE J B, BERRET J F. Cytoplasmic viscosity is a potential biomarker for metastatic breast cancer cells [J]. Nanoscale Adv, 2024, 6(6): 1727-38.

细胞生理	细胞质浓度（变化前为100%）	浓度变化原因	后果	参考文献
有丝分裂肿胀	80%～90%	未知	未知	[46]
配子成熟	增加	排水	配子抗逆性增加	[99, 100]
血细胞成熟	增加或减少；血红细胞细胞质浓度最大；单核细胞其次	血红细胞：血红蛋白大量合成	血红细胞携氧量增加	[34, 47, 101]
成骨细胞成熟	50%	细胞分化，体积增加	骨骼发育	[102]
渗透压	64%～155%	渗透压改变	微管聚合速率变化范围：64%~477%；微管解聚速率变化范围：42%~645%	[15]
翻译/降解	10%～200%	人工浓缩、稀释	翻译速度先增加后降低，降解速度增加	[13]
基因组稀释	cdc28-13突变体：923%	限制温度培养，细胞周期停滞，细胞体积增加	基因表达激活停滞；基因组稳定性降低	[78]
衰老	hTERT-RPE1细胞：590% MCF7细胞：262%	Palbociclib处理，细胞体积增加	激活 p53-p21 信号传导；53BP1不再修复 DNA 损伤；有丝分裂失败；基因组不稳定性；细胞周期永久退出	[95]

细胞生理	细胞质浓度（变化前为100%）	浓度变化原因	后果	参考文献
有丝分裂肿胀	80%～90%	未知	未知	[46]
配子成熟	增加	排水	配子抗逆性增加	[99, 100]
血细胞成熟	增加或减少；血红细胞细胞质浓度最大；单核细胞其次	血红细胞：血红蛋白大量合成	血红细胞携氧量增加	[34, 47, 101]
成骨细胞成熟	50%	细胞分化，体积增加	骨骼发育	[102]
渗透压	64%～155%	渗透压改变	微管聚合速率变化范围：64%~477%；微管解聚速率变化范围：42%~645%	[15]
翻译/降解	10%～200%	人工浓缩、稀释	翻译速度先增加后降低，降解速度增加	[13]
基因组稀释	cdc28-13突变体：923%	限制温度培养，细胞周期停滞，细胞体积增加	基因表达激活停滞；基因组稳定性降低	[78]
衰老	hTERT-RPE1细胞：590% MCF7细胞：262%	Palbociclib处理，细胞体积增加	激活 p53-p21 信号传导；53BP1不再修复 DNA 损伤；有丝分裂失败；基因组不稳定性；细胞周期永久退出	[95]

[1]	Xiangshi LIU, Yilu WU, Peng ZHAN, Tianhao HUANG, Di CAI, Peiyong QIN. State-of-the-art for alcohol dehydrogenase development and the prospect of its applications in bio-based furan compounds valorization [J]. Synthetic Biology Journal, 2023, 4(6): 1122-1139.
[2]	Wanqiu LIU, Xiangyang JI, Huiling XU, Yicong LU, Jian LI. Cell-free protein synthesis system enables rapid and efficient biosynthesis of restriction endonucleases [J]. Synthetic Biology Journal, 2023, 4(4): 840-851.
[3]	Shiming TANG, Jiyuan HU, Suiping ZHENG, Shuangyan HAN, Ying LIN. Designing, building and rapid prototyping of biosynthesis module based on cell-free system [J]. Synthetic Biology Journal, 2022, 3(6): 1250-1261.
[4]	Jiaqi HOU, Nan JIANG, Lianju MA, Yuan LU. Cell-free protein synthesis: from basic research to engineering applications [J]. Synthetic Biology Journal, 2022, 3(3): 465-486.
[5]	Xinyu YANG, Tong ZHU, Ruifeng LI, Bian WU. Enzymatic ligation technologies for the synthesis of pharmaceutical peptides and proteins [J]. Synthetic Biology Journal, 2021, 2(1): 33-45.