Transformation properties and microstructure of sputter-deposited Ni-Ti shape memory alloy thin films

The influence of annealing parameters on the martensitic phase transformation in sputter-deposited Ti rich Ni-Ti films is systematically studied by differential scanning calorimetry and by transmission electron microscopy. The annealing temperature range extends from the crystallization temperature of the films up to 900°C. For increasing temperature, multiple phase transformations, transformations via an R-phase or direct martensite/austenite transformations are observed. A similar behavior is found for increasing annealing time. Related changes of the film microstructure, such as the strongly varying distribution of round Ti2Ni precipitates in the grains, are analyzed. Transformation temperatures could be shifted over a wide range by adjusting the film composition from 48 to 54 at.% Ti. The corresponding transformation curves, grain structure as well as nature and amount of precipitates were investigated. No subsequent annealing process is required for films deposited on substrates heated above about 500°C. In this case, the as-deposited films have a very fine-grained and homogeneous microstructur

Deconstructing sarcomeric structure-function relations in titin-BioID knock-in mice

Proximity proteomics has greatly advanced the analysis of native protein complexes and subcellular structures in culture, but has not been amenable to study development and disease in vivo. Here, we have generated a knock-in mouse with the biotin ligase (BioID) inserted at titin's Z-disc region to identify protein networks that connect the sarcomere to signal transduction and metabolism. Our census of the sarcomeric proteome from neonatal to adult heart and quadriceps reveals how perinatal signaling, protein homeostasis and the shift to adult energy metabolism shape the properties of striated muscle cells. Mapping biotinylation sites to sarcomere structures refines our understanding of myofilament dynamics and supports the hypothesis that myosin filaments penetrate Z-discs to dampen contraction. Extending this proof of concept study to BioID fusion proteins generated with Crispr/CAS9 in animal models recapitulating human pathology will facilitate the future analysis of molecular machines and signaling hubs in physiological, pharmacological, and disease context

Deleting titin's C-terminal PEVK exons increases passive stiffness, alters splicing, and induces cross-sectional and longitudinal hypertrophy in skeletal muscle

The Proline, Glutamate, Valine and Lysine-rich (PEVK) region of titin constitutes an entropic spring that provides passive tension to striated muscle. To study the functional and structural repercussions of a small reduction in the size of the PEVK region, we investigated skeletal muscles of a mouse with the constitutively expressed C-terminal PEVK exons 219–225 deleted, the Ttn(Δ219–225) model (MGI: Ttn(TM 2.1Mgot)). Based on this deletion, passive tension in skeletal muscle was predicted to be increased by ∼17% (sarcomere length 3.0 μm). In contrast, measured passive tension (sarcomere length 3.0 μm) in both soleus and EDL muscles was increased 53 ± 11% and 62 ± 4%, respectively. This unexpected increase was due to changes in titin, not to alterations in the extracellular matrix, and is likely caused by co-expression of two titin isoforms in Ttn(Δ219–225) muscles: a larger isoform that represents the Ttn(Δ219–225) N2A titin and a smaller isoform, referred to as N2A2. N2A2 represents a splicing adaption with reduced expression of spring element exons, as determined by titin exon microarray analysis. Maximal tetanic tension was increased in Ttn(Δ219–225) soleus muscle (WT 240 ± 9; Ttn(Δ219–225) 276 ± 17 mN/mm2), but was reduced in EDL muscle (WT 315 ± 9; Ttn(Δ219–225) 280 ± 14 mN/mm2). The changes in active tension coincided with a switch toward slow fiber types and, unexpectedly, faster kinetics of tension generation and relaxation. Functional overload (FO; ablation) and hindlimb suspension (HS; unloading) experiments were also conducted. Ttn(Δ219–225) mice showed increases in both longitudinal hypertrophy (increased number of sarcomeres in series) and cross-sectional hypertrophy (increased number of sarcomeres in parallel) in response to FO and attenuated cross-sectional atrophy in response to HS. In summary, slow- and fast-twitch muscles in a mouse model devoid of titin's PEVK exons 219–225 have high passive tension, due in part to alterations elsewhere in splicing of titin’s spring region, increased kinetics of tension generation and relaxation, and altered trophic responses to both functional overload and unloading. This implicates titin’s C-terminal PEVK region in regulating passive and active muscle mechanics and muscle plasticity

A single-cell RNA labeling strategy for measuring stress response upon tissue dissociation

Tissue dissociation, a crucial step in single-cell sample preparation, can alter the transcriptional state of a sample through the intrinsic cellular stress response. Here we demonstrate a general approach for measuring transcriptional response during sample preparation. In our method, transcripts made during dissociation are labeled for later identification upon sequencing. We found general as well as cell-type-specific dissociation response programs in zebrafish larvae, and we observed sample-to-sample variation in the dissociation response of mouse cardiomyocytes despite well-controlled experimental conditions. Finally, we showed that dissociation of the mouse hippocampus can lead to the artificial activation of microglia. In summary, our approach facilitates experimental optimization of dissociation procedures as well as computational removal of transcriptional perturbation response