Trypanosoma brucei histone H1 inhibits RNA polymerase I transcription and is important for parasite fitness in vivo.

Trypanosoma brucei is a unicellular parasite that causes sleeping sickness in humans. Most of its transcription is constitutive and driven by RNA polymerase II. RNA polymerase I (Pol I) transcribes not only ribosomal RNA genes, but also protein-encoding genes, including variant surface glycoproteins (VSGs) and procyclins. In T. brucei, histone H1 (H1) is required for VSG silencing and chromatin condensation. However, whether H1 has a genome-wide role in transcription is unknown. Here, using RNA sequencing we show that H1 depletion changes the expression of a specific cohort of genes. Interestingly, the predominant effect is partial loss of silencing of Pol I loci, such as VSG and procyclin genes. Labelling of nascent transcripts with 4-thiouridine showed that H1 depletion does not alter the level of labelled Pol II transcripts. In contrast, the levels of 4sU-labelled Pol I transcripts were increased by two- to sixfold, suggesting that H1 preferentially blocks transcription at Pol I loci. Finally, we observed that parasites depleted of H1 grow almost normally in culture but they have a reduced fitness in mice, suggesting that H1 is important for host-pathogen interactions.

Muscle repair after physiological damage relies on nuclear migration for cellular reconstruction.

Regeneration of skeletal muscle is a highly synchronized process that requires muscle stem cells (satellite cells). We found that localized injuries, as experienced through exercise, activate a myofiber self-repair mechanism that is independent of satellite cells in mice and humans. Mouse muscle injury triggers a signaling cascade involving calcium, Cdc42, and phosphokinase C that attracts myonuclei to the damaged site via microtubules and dynein. These nuclear movements accelerate sarcomere repair and locally deliver messenger RNA (mRNA) for cellular reconstruction. Myofiber self-repair is a cell-autonomous protective mechanism and represents an alternative model for understanding the restoration of muscle architecture in health and disease.

Interrogating RNA and protein spatial subcellular distribution in smFISH data with DypFISH.

Advances in single-cell RNA sequencing have allowed for the identification of cellular subtypes on the basis of quantification of the number of transcripts in each cell. However, cells might also differ in the spatial distribution of molecules, including RNAs. Here, we present DypFISH, an approach to quantitatively investigate the subcellular localization of RNA and protein. We introduce a range of analytical techniques to interrogate single-molecule RNA fluorescence in situ hybridization (smFISH) data in combination with protein immunolabeling. DypFISH is suited to study patterns of clustering of molecules, the association of mRNA-protein subcellular localization with microtubule organizing center orientation, and interdependence of mRNA-protein spatial distributions. We showcase how our analytical tools can achieve biological insights by utilizing cell micropatterning to constrain cellular architecture, which leads to reduction in subcellular mRNA distribution variation, allowing for the characterization of their localization patterns. Furthermore, we show that our method can be applied to physiological systems such as skeletal muscle fibers.

An In Vitro System to Measure the Positioning, Stiffness, and Rupture of the Nucleus in Skeletal Muscle.

Nuclear positioning plays important roles for certain cellular functions. This is particularly relevant in skeletal muscle cells also known as myofibers in which nuclear positioning defects were shown to hinder muscle function. Myofibers are multinucleated cells with nuclei equally distributed at the periphery of the cell. However, nuclei can be found centrally located during myogenesis before anchoring at the periphery or in certain muscle disorders, either due to regenerating myofibers or defects in nuclear movement. As such, nuclear localization in myofibers (central or peripheral) can be used to assess myofiber maturity, regeneration, or health. To study how nuclei reach the periphery of myofibers during development, we devised a unique protocol to mature myofibers thereby recapitulating later stages of differentiation, including nuclear movement to the periphery. Here we describe how to use this system to study nuclear positioning and other nuclear characteristics such as nuclear stiffness or rupture.

In Vitro Differentiation of Mature Myofibers for Live Imaging.

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily conserved structures that compose it are assembled remains under investigation. Their size and physiological features often constrain manipulation and imaging applications. The culture of immortalized cell lines is widely used, but it can only replicate the early steps of differentiation. Here, we describe a protocol that enables easy genetic manipulation of myofibers originating from primary mouse myoblasts. After one week of differentiation, the myofibers display contractility, aligned sarcomeres and triads, as well as peripheral nuclei. The entire differentiation process can be followed by live imaging or immunofluorescence. This system combines the advantages of the existing ex vivo and in vitro protocols. The possibility of easy and efficient transfection as well as the ease of access to all differentiation stages broadens the potential applications. Myofibers can subsequently be used not only to address relevant developmental and cell biology questions, but also to reproduce muscle disease phenotypes for clinical applications.