Enhancement of Biological Reactions on Cell Surfaces via Macromolecular Crowding

Chapanian, Rafi; Kwan, David H.; Constantinescu, Iren; Shaikh, Fathima A.; Rossi, Nicholas A.A.; Withers, Stephen G; Kizhakkedathu, Jayachandran N

doi:10.1038/ncomms5683

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Enhancement of Biological Reactions on Cell Surfaces via Macromolecular Crowding

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Author(s): Rafi Chapanian ¹ ^, ² , David H. Kwan ³ ^, ⁵ , Iren Constantinescu ¹ , Fathima A. Shaikh ³ , Nicholas A.A. Rossi ¹ , Stephen G. Withers ³ ^, ⁴ ^, ⁵ , Jayachandran N. Kizhakkedathu ¹ ^, ² ^, ³

Publication date (Electronic): 20 August 2014

Journal: Nature communications

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Abstract

The reaction of macromolecules such as enzymes and antibodies with cell surfaces is often an inefficient process, requiring large amounts of expensive reagent. Here we report a general method based on macromolecular crowding with a range of neutral polymers to enhance such reactions, using red blood cells (RBCs) as a model system. Rates of conversion of Type A and B red blood cells to universal O type by removal of antigenic carbohydrates with selective glycosidases are increased up to 400-fold in the presence of crowders. Similar enhancements are seen for antibody binding. We further explore the factors underlying these enhancements using confocal microscopy and fluorescent recovery after bleaching (FRAP) techniques with various fluorescent protein fusion partners. Increased cell-surface concentration due to volume exclusion, along with two-dimensionally confined diffusion of enzymes close to the cell surface, appear to be the major contributing factors.

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Anomalous diffusion of proteins due to molecular crowding.

Daniel S. Banks, Cécile Fradin (2005)

We have studied the diffusion of tracer proteins in highly concentrated random-coil polymer and globular protein solutions imitating the crowded conditions encountered in cellular environments. Using fluorescence correlation spectroscopy, we measured the anomalous diffusion exponent alpha characterizing the dependence of the mean-square displacement of the tracer proteins on time, r(2)(t) approximately t(alpha). We observed that the diffusion of proteins in dextran solutions with concentrations up to 400 g/l is subdiffusive (alpha < 1) even at low obstacle concentration. The anomalous diffusion exponent alpha decreases continuously with increasing obstacle concentration and molecular weight, but does not depend on buffer ionic strength, and neither does it depend strongly on solution temperature. At very high random-coil polymer concentrations, alpha reaches a limit value of alpha(l) approximately 3/4, which we take to be the signature of a coupling between the motions of the tracer proteins and the segments of the dextran chains. A similar, although less pronounced, subdiffusive behavior is observed for the diffusion of streptavidin in concentrated globular protein solutions. These observations indicate that protein diffusion in the cell cytoplasm and nucleus should be anomalous as well, with consequences for measurements of solute diffusion coefficients in cells and for the modeling of cellular processes relying on diffusion.

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Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion.

R. Swaminathan, C.P. Hoang, A.S. Verkman (1997)

The green fluorescent protein (GFP) was used as a noninvasive probe to quantify the rheological properties of cell cytoplasm. GFP mutant S65T was purified from recombinant bacteria for solution studies, and expressed in CHO cell cytoplasm. GFP-S65T was brightly fluorescent in solution (lambda ex 492 nm, lambda em 509 nm) with a lifetime of 2.9 ns and a rotational correlation time (tc) of 20 ns. Recovery of GFP fluorescence after photobleaching was complete with a half-time (t1/2) in aqueous saline of 30 +/- 2 ms (5-micron diameter spot), giving a diffusion coefficient of 8.7 x 10(-7) cm2/s. The t1/2 was proportional to solution viscosity and was dependent on spot diameter. In contrast to fluorescein. GFP photobleaching efficiency was not affected by solution O2 content, triplet state quenchers, singlet oxygen scavengers, and general radical quenchers. In solutions of higher viscosity, an additional, rapid GFP recovery process was detected and ascribed to reversible photobleaching. The t1/2 for reversible photobleaching was 1.5-5.5 ms (relative viscosity 5-250), was independent of spot diameter, and was unaffected by O2 or quenchers. In cell cytoplasm, time-resolved microfluorimetry indicated a GFP lifetime of 2.6 ns and a tc of 36 +/- 3 ns, giving a relative viscosity (cytoplasm versus water) of 1.5. Photobleaching recovery of GFP in cytoplasm was 82 +/- 2% complete with a t1/2 of 83 +/- 6 ms, giving a relative viscosity of 3.2. GFP translational diffusion increased 4.7-fold as cells swelled from a relative volume of 0.5 to 2. Taken together with measurements of GFP translation and rotation in aqueous dextran solutions, the data in cytoplasm support the view that the primary barrier to GFP diffusion is collisional interactions between GFP and macromolecular solutes.

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Molecular crowding shapes gene expression in synthetic cellular nanosystems

Cheemeng Tan, Saumya Saurabh, Marcel Bruchez … (2013)

Summary The integration of synthetic and cell-free biology has made tremendous strides towards creating artificial cellular nanosystems using concepts from solution-based chemistry: only the concentrations of reacting species modulate gene expression rates. However, it is known that macromolecular crowding, a key feature of natural cells, can dramatically influence biochemical kinetics by volume exclusion effects that reduce diffusion rates and enhance binding rates of macromolecules. Here, we demonstrate that macromolecular crowding can increase the robustness of gene expression through integrating synthetic cellular components of biological circuits and artificial cellular nanosystems. In addition, we reveal how ubiquitous cellular modules, including genetic components, a negative feedback loop, and the size of crowding molecules, can fine tune gene circuit response to molecular crowding. By bridging a key gap between artificial and living cells, our work has implications for efficient and robust control of both synthetic and natural cellular circuits.

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Author and article information

Journal

Journal ID (nlm-journal-id): 101528555

Journal ID (pubmed-jr-id): 37539

Journal ID (nlm-ta): Nat Commun

Journal ID (iso-abbrev): Nat Commun

Title: Nature communications

ISSN (Electronic): 2041-1723

Publication date Nihms-submitted: 3 August 2016

Publication date (Electronic): 20 August 2014

Publication date Collection: 2014

Publication date PMC-release: 09 August 2016

Volume: 5

Page: 4683

Affiliations

[1 ]Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada, V6T 1Z3

[2 ]Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada, V6T 1Z3

[3 ]Department of Chemistry, University of British Columbia, Vancouver, BC, Canada, V6T 1Z3

[4 ]Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada, V6T 1Z3

[5 ]Centre for High-Throughput Biology, University of British Columbia, Vancouver, BC, Canada, V6T 1Z3

Author notes

[* ]Corresponding authors: Dr. Stephen G Withers FRS Khorana Professor, Department of Chemistry and Biochemistry, University of British Columbia, 2036 Main Mall, Vancouver, B.C., Canada V6T 1Z1, Tel 604 822 3402, withers@ 123456chem.ubc.ca . Or Dr. Jayachandran N Kizhakkedathu, Department of Pathology and Lab Medicine, Center for Blood Research, Department of Chemistry, University of British Columbia, 2350 Health Sciences Mall, Life Sciences Centre, Vancouver, BC, CANADA V6T 1Z3, Phone: 604-822-7085, Fax: 604-822-7742, jay@ 123456pathology.ubc.ca

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Manuscript ID: CAMS4560

DOI: 10.1038/ncomms5683

PMC ID: 4978540

PubMed ID: 25140641

SO-VID: 141f60ef-1ec5-49ec-837e-68a7639a19c9

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Enhancement of Biological Reactions on Cell Surfaces via Macromolecular Crowding

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Most cited references 49

Anomalous diffusion of proteins due to molecular crowding.

Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion.

Molecular crowding shapes gene expression in synthetic cellular nanosystems

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