scholarly journals Rapid extraction and kinetic analysis of protein complexes from single cells

2021 ◽  
Author(s):  
Sena Sarıkaya ◽  
Daniel J Dickinson
Keyword(s):  
Single Molecule ◽  
Protein Complex ◽  
Cell Biology ◽  
Dynamic Range ◽  
Protein Complexes ◽  
Single Cells ◽  
Cell Lysis ◽  
Cellular Protein ◽  
In Vitro Assays

Proteins contribute to cell biology by forming dynamic, regulated interactions, and measuring these interactions is a foundational approach in biochemistry. We present a rapid, quantitative in vivo assay for protein-protein interactions, based on optical cell lysis followed by time-resolved single-molecule analysis of protein complex binding to an antibody-coated substrate. We show that our approach has better reproducibility, higher dynamic range, and lower background than previous single-molecule pull-down assays. Furthermore, we demonstrate that by monitoring cellular protein complexes over time after cell lysis, we can measure the dissociation rate constant of a cellular protein complex, providing information about binding affinity and kinetics. Our dynamic single-cell, single-molecule pull-down method thus approaches the biochemical precision that is often sought from in vitro assays, while being applicable to native protein complexes isolated from single cells in vivo.

Assembly and Dynamics of the Bacterial Flagellum

2020 ◽  
Vol 74 (1) ◽  
pp. 181-200 ◽  
Author(s):  
Judith P. Armitage ◽  
Richard M. Berry
Keyword(s):  
Single Molecule ◽  
Protein Complexes ◽  
Complex Structure ◽  
Live Cells ◽  
Flagellar Motor ◽  
Interacting Protein ◽  
Bacterial Flagellum ◽  
Bacterial Flagellar Motor ◽  
The Stability

The bacterial flagellar motor is the most complex structure in the bacterial cell, driving the ion-driven rotation of the helical flagellum. The ordered expression of the regulon and the assembly of the series of interacting protein rings, spanning the inner and outer membranes to form the ∼45–50-nm protein complex, have made investigation of the structure and mechanism a major challenge since its recognition as a rotating nanomachine about 40 years ago. Painstaking molecular genetics, biochemistry, and electron microscopy revealed a tiny electric motor spinning in the bacterial membrane. Over the last decade, new single-molecule and in vivo biophysical methods have allowed investigation of the stability of this and other large protein complexes, working in their natural environment inside live cells. This has revealed that in the bacterial flagellar motor, protein molecules in both the rotor and stator exchange with freely circulating pools of spares on a timescale of minutes, even while motors are continuously rotating. This constant exchange has allowed the evolution of modified components allowing bacteria to keep swimming as the viscosity or the ion composition of the outside environment changes.

CD38-Targeted Immunochemotherapy Of Multiple Myeloma: Preclinical Evidence For Its Combinatorial Use In Lenalidomide and Bortezomib Refractory/Intolerant MM Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 277-277 ◽  
Author(s):  
Inger S. Nijhof ◽  
Willy A. Noort ◽  
Jeroen Lammerts van Bueren ◽  
Berris van Kessel ◽  
Joost M. Bakker ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Research Funding ◽  
Plasma Cells ◽  
Cell Lysis ◽  
In Vitro Assays ◽  
Human Antibody ◽  
Myeloma Cells ◽  
Clinical Phase

Abstract Multiple myeloma (MM) remains an incurable malignancy of clonal plasma cells. Although the new generation of immunomodulatory agents, such as lenalidomide (LEN), and the potent proteasome inhibitor bortezomib (BORT) have significantly improved the overall survival of MM patients, all chemotherapy strategies are eventually hampered by the development of drug-resistance. The outcome of patients who are refractory to thalidomide, lenalidomide (LEN) and bortezomib (BORT) is very poor. Set out with the idea that targeted immunotherapy with human antibodies may offer new perspectives for MM patients, we have recently developed daratumumab (DARA), a CD38 human antibody with broad-spectrum killing activity, mainly via ADCC (antibody dependent cellular cytotoxicity) and CDC (complement dependent cytotoxicity). In our previous preclinical studies and in current clinical phase I/II trials, DARA induces marked anti-MM activity. Based on these encouraging results, we now explored the potential activity of DARA for patients who are refractory to LEN- and/or BORT. In a recently developed human-mouse hybrid model that allows the in vivo engraftment and outgrowth of patient-derived primary myeloma cells in immune deficient Rag2-/-gc-/- mice, single dose DARA treatment appeared to effectively inhibit the malignant expansion of primary MM cells derived from a LEN- and BORT-refractory patient, indicating the potential efficacy of DARA even in LEN/BORT refractory patients. To substantiate the conclusions of these in vivo data, we conducted in vitro assays, in which full BM-MNCs from LEN (n=11) and LEN/BORT (n=8) refractory patients were treated with DARA alone or the combination of DARA with LEN or BORT to induce MM cell lysis. As expected, LEN alone induced no or little lysis of MM cells in the LEN-refractory patients and also BORT was not able to induce any lysis in the BORT-refractory patients. On the contrary, DARA induced substantial levels of MM cell lysis in all LEN and LEN/BORT-refractory patients. This lysis was significantly enhanced by combination with LEN or BORT. The combination of DARA and BORT improved MM lysis by additive mechanisms. However, LEN improved DARA-mediated lysis of MM cells in a synergistic manner through the activation of effector cells involved in DARA-mediated ADCC. In conclusion, our results demonstrate that DARA is also effective against multiple myeloma cells derived from LEN- and BORT-refractory patients. Especially LEN seems to improve responses in a synergistic manner. Our results provide a rationale for clinical evaluation of DARA in combination with LEN to achieve more effective results in LEN- and BORT-refractory patients. Disclosures: Lammerts van Bueren: Genmab: Employment. Bakker:Genmab: Employment. Parren:Genmab: Employment. van de Donk:Celgene: Research Funding. Lokhorst:Genmab A/S: Consultancy, Research Funding; Celgene: Honoraria; Johnson-Cilag: Honoraria; Mudipharma: Honoraria.

scholarly journals Watching cellular machinery in action, one molecule at a time

2016 ◽  
Vol 216 (1) ◽  
pp. 41-51 ◽  
Author(s):  
Enrico Monachino ◽  
Lisanne M. Spenkelink ◽  
Antoine M. van Oijen
Keyword(s):  
Dynamic Behavior ◽  
Single Molecule ◽  
Protein Complexes ◽  
Imaging Techniques ◽  
Ensemble Averaging ◽  
Cellular Processes ◽  
Cellular Machinery

Single-molecule manipulation and imaging techniques have become important elements of the biologist’s toolkit to gain mechanistic insights into cellular processes. By removing ensemble averaging, single-molecule methods provide unique access to the dynamic behavior of biomolecules. Recently, the use of these approaches has expanded to the study of complex multiprotein systems and has enabled detailed characterization of the behavior of individual molecules inside living cells. In this review, we provide an overview of the various force- and fluorescence-based single-molecule methods with applications both in vitro and in vivo, highlighting these advances by describing their applications in studies on cytoskeletal motors and DNA replication. We also discuss how single-molecule approaches have increased our understanding of the dynamic behavior of complex multiprotein systems. These methods have shown that the behavior of multicomponent protein complexes is highly stochastic and less linear and deterministic than previously thought. Further development of single-molecule tools will help to elucidate the molecular dynamics of these complex systems both inside the cell and in solutions with purified components.

scholarly journals From isolated light-harvesting complexes to the thylakoid membrane: a single-molecule perspective

Nanophotonics ◽  
2018 ◽  
Vol 7 (1) ◽  
pp. 81-92 ◽  
Author(s):  
J. Michael Gruber ◽  
Pavel Malý ◽  
Tjaart P.J. Krüger ◽  
Rienk van Grondelle
Keyword(s):  
Single Molecule ◽  
Thylakoid Membrane ◽  
Light Harvesting ◽  
Protein Complexes ◽  
Conformational Dynamics ◽  
Physical Models ◽  
Photochemical Quenching ◽  
Light Harvesting Complexes ◽  
Fluorescence Measurements

AbstractThe conversion of solar radiation to chemical energy in plants and green algae takes place in the thylakoid membrane. This amphiphilic environment hosts a complex arrangement of light-harvesting pigment-protein complexes that absorb light and transfer the excitation energy to photochemically active reaction centers. This efficient light-harvesting capacity is moreover tightly regulated by a photoprotective mechanism called non-photochemical quenching to avoid the stress-induced destruction of the catalytic reaction center. In this review we provide an overview of single-molecule fluorescence measurements on plant light-harvesting complexes (LHCs) of varying sizes with the aim of bridging the gap between the smallest isolated complexes, which have been well-characterized, and the native photosystem. The smallest complexes contain only a small number (10–20) of interacting chlorophylls, while the native photosystem contains dozens of protein subunits and many hundreds of connected pigments. We discuss the functional significance of conformational dynamics, the lipid environment, and the structural arrangement of this fascinating nano-machinery. The described experimental results can be utilized to build mathematical-physical models in a bottom-up approach, which can then be tested on larger in vivo systems. The results also clearly showcase the general property of biological systems to utilize the same system properties for different purposes. In this case it is the regulated conformational flexibility that allows LHCs to switch between efficient light-harvesting and a photoprotective function.

RNA and RNA–Protein Complex Crystallography and its Challenges

2014 ◽  
Vol 67 (12) ◽  
pp. 1741 ◽  
Author(s):  
Janine K. Flores ◽  
James L. Walshe ◽  
Sandro F. Ataide
Keyword(s):  
Protein Complex ◽  
Protein Complexes ◽  
Future Directions ◽  
Vast Number ◽  
X Ray Crystallography ◽  
Rna Biology ◽  
Non Coding Rnas ◽  
Rna Protein Complexes

RNA biology has changed completely in the past decade with the discovery of non-coding RNAs. Unfortunately, obtaining mechanistic information about these RNAs alone or in cellular complexes with proteins has been a major problem. X-ray crystallography of RNA and RNA–protein complexes has suffered from the major problems encountered in preparing and purifying them in large quantity. Here, we review the available techniques and methods in vitro and in vivo used to prepare and purify RNA and RNA–protein complex for crystallographic studies. We also discuss the future directions necessary to explore the vast number of RNA species waiting for their atomic-resolution structure to be determined.

scholarly journals The Min-protein oscillations in Escherichia coli : an example of self-organized cellular protein waves

2018 ◽  
Vol 373 (1747) ◽  
pp. 20170111 ◽  
Author(s):  
Lukas Wettmann ◽  
Karsten Kruse
Keyword(s):  
Escherichia Coli ◽  
Cell Biology ◽  
Experimental Studies ◽  
Cellular Protein ◽  
Self Organization ◽  
Division Site ◽  
Min Proteins ◽  
Spatio Temporal

In the rod-shaped bacterium Escherichia coli , selection of the cell centre as the division site involves pole-to-pole oscillations of the proteins MinC, MinD and MinE. This spatio-temporal pattern emerges from interactions among the Min proteins and with the cytoplasmic membrane. Combining experimental studies in vivo and in vitro together with theoretical analysis has led to a fairly good understanding of Min-protein self-organization. In different geometries, the system can, in addition to standing waves, also produce travelling planar and spiral waves as well as coexisting stable stationary distributions. Today it stands as one of the best-studied examples of cellular self-organization of proteins. This article is part of the theme issue ‘Self-organization in cell biology’.

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