ABSTRACT
Filopodia are actin-rich structures, present on the surface of eukaryotic cells. These structures play a pivotal role by allowing cells to explore their environment, generate mechanical forces or perform chemical signaling. Their complex dynamics includes buckling, pulling, length and shape changes. We show that filopodia additionally explore their 3D extracellular space by combining growth and shrinking with axial twisting and buckling. Importantly, the actin core inside filopodia performs a twisting or spinning motion which is observed for a range of cell types spanning from earliest development to highly differentiated tissue cells. Non-equilibrium physical modeling of actin and myosin confirm that twist is an emergent phenomenon of active filaments confined in a narrow channel which is supported by measured traction forces and helical buckles that can be ascribed to accumulation of sufficient twist. These results lead us to conclude that activity induced twisting of the actin shaft is a general mechanism underlying fundamental functions of filopodia.
Subject(s)
Actins , Pseudopodia , Actin Cytoskeleton/metabolism , Actins/metabolism , Motion , Myosins/metabolism , Pseudopodia/metabolismABSTRACT
BRCA2 is a key breast cancer associated protein that is predicted to have interspersed regions of intrinsic disorder. Intrinsic disorder coupled with large size likely allows BRCA2 to sample a broad range of conformational space. We expect that the resulting dynamic arrangements of BRCA2 domains are a functionally important aspect of its role in homologous recombination DNA repair. To determine the architectural organization and the associated conformational landscape of BRCA2, we used scanning force microscopy based single molecule analyses to map the flexible regions of the protein and characterize which regions influence oligomerization. We show that the N- and the C-terminal regions are the main flexible regions. Both of these regions also influence BRCA2 oligomerization and interaction with RAD51. In the central Brc repeat region, Brc 1-4 and Brc 5-8 contribute synergistically to BRCA2 interaction with RAD51. We also analysed several single amino acid changes that are potentially clinically relevant and found one, the variant of F1524V, which disrupts key interactions and alters the conformational landscape of the protein. We describe the overall conformation spectrum of BRCA2, which suggests that dynamic structural transitions are key features of its biological function, maintaining genomic stability.
Subject(s)
BRCA2 Protein/chemistry , BRCA2 Protein/metabolism , Rad51 Recombinase/metabolism , BRCA2 Protein/genetics , Humans , Microscopy, Atomic Force , Mutation, Missense , Protein Conformation , Protein Interaction Domains and Motifs , Protein Multimerization , Rad51 Recombinase/geneticsABSTRACT
During mRNA translation, the genetic information stored in mRNA is translated into a protein sequence. It is imperative that the genetic information is translated with high precision. Surprisingly, however, recent experimental evidence has demonstrated that translation can be highly heterogeneous, even among different mRNA molecules derived from a single gene in an individual cell; multiple different polypeptides can be produced from a single mRNA molecule and the rate of translation can vary in both space and time. However, whether translational heterogeneity serves an important cellular function, or rather predominantly represents gene expression 'noise' remains an open question. In this review, we discuss the molecular basis and potential functions of such translational heterogeneity.
Subject(s)
Protein Biosynthesis/genetics , RNA, Messenger/genetics , Amino Acid Sequence , Animals , Humans , Models, Biological , RNA, Messenger/chemistry , RNA, Messenger/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Ribosomes/metabolismABSTRACT
mRNA translation is a key step in gene expression. Proper regulation of translation efficiency ensures correct protein expression levels in the cell, which is essential to cell function. Different methods used to study translational control in the cell rely on population-based assays that do not provide information about translational heterogeneity between cells or between mRNAs of the same gene within a cell, and generally provide only a snapshot of translation. To study translational heterogeneity and measure translation dynamics, we have developed microscopy-based methods that enable visualization of translation of single mRNAs in live cells. These methods consist of a set of genetic tools, an imaging-based approach and sophisticated computational tools. Using the translation imaging method, one can investigate many new aspects of translation in single living cells, such as translation start-site selection, 3'-UTR (untranslated region) translation and translation-coupled mRNA decay. Here, we describe in detail how to perform such experiments, including reporter design, cell line generation, image acquisition and analysis. This protocol also provides a detailed description of the image analysis pipeline and computational modeling that will enable non-experts to correctly interpret fluorescence measurements. The protocol takes 2-4 d to complete (after cell lines expressing all required transgenes have been generated).
Subject(s)
Image Processing, Computer-Assisted/methods , Protein Biosynthesis/genetics , RNA, Messenger/analysis , Single Molecule Imaging/methods , HEK293 Cells , Humans , RNA, Messenger/geneticsABSTRACT
mRNA translation is a key step in decoding genetic information. Genetic decoding is surprisingly heterogeneous because multiple distinct polypeptides can be synthesized from a single mRNA sequence. To study translational heterogeneity, we developed the MoonTag, a fluorescence labeling system to visualize translation of single mRNAs. When combined with the orthogonal SunTag system, the MoonTag enables dual readouts of translation, greatly expanding the possibilities to interrogate complex translational heterogeneity. By placing MoonTag and SunTag sequences in different translation reading frames, each driven by distinct translation start sites, start site selection of individual ribosomes can be visualized in real time. We find that start site selection is largely stochastic but that the probability of using a particular start site differs among mRNA molecules and can be dynamically regulated over time. This study provides key insights into translation start site selection heterogeneity and provides a powerful toolbox to visualize complex translation dynamics.
Subject(s)
Fluorescent Dyes/chemistry , RNA, Messenger/metabolism , Single Molecule Imaging/methods , 3' Untranslated Regions , 5' Untranslated Regions , Cell Line, Tumor , Genes, Reporter , HEK293 Cells , Humans , Peptide Chain Initiation, Translational , RNA, Messenger/chemistry , Ribosomes/metabolism , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/immunologyABSTRACT
Nonsense-mediated decay (NMD) is a surveillance system that degrades mRNAs containing a premature termination codon (PTC) and plays important roles in protein homeostasis and disease. The efficiency of NMD is variable, impacting the clinical outcome of genetic mutations. However, limited resolution of bulk analyses has hampered the study of NMD efficiency. Here, we develop an assay to visualize NMD of individual mRNA molecules in real time. We find that NMD occurs with equal probability during each round of translation of an mRNA molecule. However, this probability is variable and depends on the exon sequence downstream of the PTC, the PTC-to-intron distance, and the number of introns both upstream and downstream of the PTC. Additionally, a subpopulation of mRNAs can escape NMD, further contributing to variation in NMD efficiency. Our study uncovers real-time dynamics of NMD, reveals key mechanisms that influence NMD efficiency, and provides a powerful method to study NMD.
