Cryo-electron tomography reveals the structural diversity of cardiac proteins in their cellular context.

Cardiovascular diseases are a leading cause of death worldwide, but our understanding of the underlying mechanisms is limited, in part because of the complexity of the cellular machinery that controls the heart muscle contraction cycle. Cryogenic electron tomography (cryo-ET) provides a way to visualize diverse cellular machinery while preserving contextual information like subcellular localization and transient complex formation, but this approach has not been widely applied to the study of heart muscle cells (cardiomyocytes). Here, we deploy a platform for studying cardiovascular disease by combining cryo-ET with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). After developing a cryo-ET workflow for visualizing macromolecules in hiPSC-CMs, we reconstructed sub-nanometer resolution structures of the human thin filament, a central component of the contractile machinery. We also visualized a previously unobserved organization of a regulatory complex that connects muscle contraction to calcium signaling (the troponin complex), highlighting the value of our approach for interrogating the structures of cardiac proteins in their cellular context.

A missense point mutation in nerve growth factor (NGFR100W) results in selective peripheral sensory neuropathy.

The R100W mutation in nerve growth factor is associated with hereditary sensory autonomic neuropathy V in a Swedish family. These patients develop severe loss of perception to deep pain but with apparently normal cognitive functions. To better understand the disease mechanism, we examined a knockin mouse model of HSAN V. The homozygous mice showed significant structural deficits in intra-epidermal nerve fibers (IENFs) at birth. These mice had a total loss of pain perception at ∼2 months of age and often failed to survive to adulthood. Heterozygous mutant mice developed a progressive degeneration of small sensory fibers both behaviorally and functionally: they showed a progressive loss of IENFs starting at the age of 9 months accompanied with progressive loss of perception to painful stimuli such as noxious temperature. Quantitative analysis of lumbar 4/5 dorsal root ganglia revealed a significant reduction in small size neurons, while analysis of sciatic nerve fibers revealed the heterozygous mutant mice had no reduction in myelinated nerve fibers. Significantly, the amount of NGF secreted from mouse embryonic fibroblasts were reduced from both heterozygous and homozygous mice compared to their wild-type littermates. Interestingly, the heterozygous mice showed no apparent structural alteration in the brain: neither the anterior cingulate cortex nor the medial septum including NGF-dependent basal forebrain cholinergic neurons. Accordingly, these animals did not develop appreciable deficits in tests for brain function. Our study has thus demonstrated that the NGFR100W mutation likely affects the structure and function of peripheral sensory neurons.

Transcribe this way: Rap1 confers promoter directionality by repressing divergent transcription.

In eukaryotes, divergent transcription is a major source of noncoding RNAs. Recent studies have uncovered that in yeast, the transcription factor Rap1 restricts transcription in the divergent direction and thereby controls promoter directionality. Here, we summarize these findings, propose regulatory principles, and discuss the implications for eukaryotic gene regulation.

Repression of Divergent Noncoding Transcription by a Sequence-Specific Transcription Factor.

Many active eukaryotic gene promoters exhibit divergent noncoding transcription, but the mechanisms restricting expression of these transcripts are not well understood. Here, we demonstrate how a sequence-specific transcription factor represses divergent noncoding transcription at highly expressed genes in yeast. We find that depletion of the transcription factor Rap1 induces noncoding transcription in a large fraction of Rap1-regulated gene promoters. Specifically, Rap1 prevents transcription initiation at cryptic promoters near its binding sites, which is uncoupled from transcription regulation in the protein-coding direction. We further provide evidence that Rap1 acts independently of previously described chromatin-based mechanisms to repress cryptic or divergent transcription. Finally, we show that divergent transcription in the absence of Rap1 is elicited by the RSC chromatin remodeler. We propose that a sequence-specific transcription factor limits access of basal transcription machinery to regulatory elements and adjacent sequences that act as divergent cryptic promoters, thereby providing directionality toward productive transcription.