Biophysics

Immotile cilia of crown cells at the node of mouse embryos are required for sensing leftward fluid flow that gives rise to the breaking of left-right (L-R) symmetry. The flow-sensing mechanism has long remained elusive, mainly because of difficulties inherent in manipulating and precisely analyzing the cilium. Recent progress in optical microscopy and biophysical analysis has allowed us to study the mechanical signals involving primary cilia. In this study, we used high-resolution imaging with mechanical modeling to assess the membrane tension in a single cilium. Optical tweezers, a technique used to trap sub-micron-sized particles with a highly focused laser beam, allowed us to manipulate individual cilia. Super-resolution microscopy allowed us to discern the precise localization of ciliary proteins. Using this protocol, we provide a method for applying these techniques to cilia in mouse embryonic nodes. This method is widely applicable to the determination of mechanical signals in other cilia.

Single-molecule measurements provide statistical distributions of molecular properties, in addition to the ensemble averages. Evanescent detection approaches have been widely used for single-molecule detection because the evanescent field can significantly enhance the light-analyte interaction and reduce the background noise. However, current evanescent single-molecule detection systems mostly require specially designed sensing components. Here, we show that single proteins can be imaged on a plain cover glass surface by detecting the evanescent waves scattered by the target molecules. This allows us to quantify the protein–antibody interactions at the single-molecule level. This protocol describes a label-free single-molecule imaging approach with conventional consumables and may pave the road for detecting single molecules with commercial optical microscopy.

Kinetoplastids are unicellular eukaryotic parasites responsible for human pathologies such as Chagas disease, sleeping sickness or Leishmaniasis, caused by Trypanosoma cruzi, Trypanosoma brucei, and various Leishmania spp., respectively. They harbor a single large mitochondrion that is essential for the survival of the parasite. Interestingly, most of the mitochondrial gene expression machineries and processes present significant differences from their nuclear and cytosolic counterparts. A striking example concerns their mitochondrial ribosomes, in charge of translating the few essential mRNAs encoded by mitochondrial genomes. Here, we present a detailed protocol including the specific procedures to isolate mitochondria from two species of kinetoplastids, T. cruzi and L. tarentolae, by differential centrifugations. Then, we detail the protocol to purify mitochondrial ribosomal complexes from these two species of parasites (including ribosomal maturating complexes) by a sucrose gradient approach. Finally, we describe how to prepare cryo-electron microscopy (cryo-EM) grids from these two sorts of samples. This protocol will be useful for further studies aiming at analyzing mitochondrial translation regulation.

Single molecule tracking (SMT) is a powerful technique to study molecular dynamics, and is particularly adapted to monitor the motion and interactions of cell membrane components. Assessing interactions among two molecular populations is classically performed by several approaches, including dual-color videomicroscopy, which allows monitoring of interactions through colocalization events. Other techniques, such as fluorescence recovery after photobleaching (FRAP), Förster resonance energy transfer (FRET), and fluorescence correlation spectroscopy (FCS), are also utilized to measure molecular dynamics.

We developed MTT2col, a set of algorithmic tools extending multi-target tracing (MTT) to dual-color acquisition (https://github.com/arnauldserge1/MTT2col). In this protocol, we used MTT2col to monitor adhesion molecules at the contact between leukemic stem cells and stromal cells, a process involved in cancer resistance to chemotherapy and in relapse. Our dual-color single molecule protocol includes the following steps: (i) labeling molecules of interest with fluorescent probes, (ii) video-acquisition, (iii) analyses using our MTT2col in-house software, to obtain positions and trajectories, followed by (iv) detailed analyses of colocalization, distribution, and dynamic motion modes, according to the issues addressed. MTT2col is a robust and efficient SMT algorithm. Both MTT and MTT2col are open-source software that can be adapted and further developed for specific analyses.

Graphical abstract:

DNA replication always encounters numerous endogenous and exogenous stresses during the cell cycle. Measuring the cell responses to stress has primarily relied on cell survival and incorporation of radioactive dNTPs, which is limited in resolution. A higher resolution is required to monitor how replication and repair respond to these stresses. This protocol describes a procedure to monitor the length of new synthesized DNA in a single molecular resolution called DNA fiber assay. The fiber assay relies on labeling of nascent DNA with the nucleoside analog 5-Chloro-2'-deoxyuridine (CldU) and 5-Iodo-2'-deoxyuridine (IdU). We can visualize the labeled nascent DNA in single molecular resolution by immunostaining. By measuring labeled DNA length, the assay permits interrogation of replication speed at given conditions, end processing at the reversed fork, and fork restart after repair.

Electrophoresis and Western blot are important tools in protein research for detection and identification of proteins. These traditional techniques separate the proteins based on size and charge differences and identify the proteins by antibody binding. Over the past decade, the emergence of single-molecule techniques has shown great potential in improving the resolution of the traditional protein analysis methods to the single-molecule level. However, such single-molecule techniques measure either size or charge, and it is challenging to measure both at the same time. Recently, we have developed a single-molecule approach to address this problem. We tether the single proteins to a surface with a polymer linker and drive them into oscillation with an electric field. By tracking the electromechanical response of the proteins to the field using an optical imaging method, the size and charge can be obtained simultaneously. Binding of antibodies or ions to the tethered protein also changes the size and charge, which allows us to probe the interactions. This protocol includes fabrication of protein oscillators, configuration of the optical detection system, and analysis of the oscillation signal for quantification of protein size and charge. We wish this protocol will enable researchers to perform comprehensive single-protein analysis on a single platform.