Three-dimensional architecture of the rod sensory cilium and its disruption in retinal neurodegeneration.

Defects in primary cilia lead to devastating disease because of their roles in sensation and developmental signaling but much is unknown about ciliary structure and mechanisms of their formation and maintenance. We used cryo-electron tomography to obtain 3D maps of the connecting cilium and adjacent cellular structures of a modified primary cilium, the rod outer segment, from wild-type and genetically defective mice. The results reveal the molecular architecture of the cilium and provide insights into protein functions. They suggest that the ciliary rootlet is involved in cellular transport and stabilizes the axoneme. A defect in the BBSome membrane coat caused defects in vesicle targeting near the base of the cilium. Loss of the proteins encoded by the Cngb1 gene disrupted links between the disk and plasma membranes. The structures of the outer segment membranes support a model for disk morphogenesis in which basal disks are enveloped by the plasma membrane.

Two Pools of Vesicles Associated with Synaptic Ribbons Are Molecularly Prepared for Release.

Neurons that form ribbon-style synapses are specialized for continuous exocytosis. To this end, their synaptic terminals contain numerous synaptic vesicles, some of which are ribbon associated, that have difference susceptibilities for undergoing Ca2+-dependent exocytosis. In this study, we probed the relationship between previously defined vesicle populations and determined their fusion competency with respect to SNARE complex formation. We found that both the rapidly releasing vesicle pool and the releasable vesicle pool of the retinal bipolar cell are situated at the ribbon-style active zones, where they functionally interact. A peptide inhibitor of SNARE complex formation failed to block exocytosis from either pool, suggesting that these two vesicle pools have formed the SNARE complexes necessary for fusion. By contrast, a third, slower component of exocytosis was blocked by the peptide, as was the functional replenishment of vesicle pools, indicating that few vesicles outside of the ribbon-style active zones were initially fusion competent. In cone photoreceptors, similar to bipolar cells, fusion of the initial ribbon-associated synaptic vesicle cohort was not blocked by the SNARE complex-inhibiting peptide, whereas a later phase of exocytosis, attributable to the recruitment and subsequent fusion of vesicles newly arrived at the synaptic ribbons, was blocked. Together, our results support a model in which stimulus-evoked exocytosis in retinal ribbon synapses is SNARE-dependent; where vesicles higher up on the synaptic ribbon replenish the rapidly releasing vesicle pool; and at any given time, there are sufficient SNARE complexes to support the fusion of the entire ribbon-associated cohort of vesicles. An important implication of these results is that ribbon-associated vesicles can form intervesicular SNARE complexes, providing mechanistic insight into compound fusion at ribbon-style synapses.

Case report: Molecular profiling facilitates the diagnosis of a challenging case of lung cancer with choriocarcinoma features.

Accurate diagnoses are crucial in determining the most effective treatment across different cancers. In challenging cases, morphology-based traditional pathology methods have important limitations, while molecular profiling can provide valuable information to guide clinical decisions. We present a 35-year female with lung cancer with choriocarcinoma features. Her disease involved the right lower lung, brain, and thoracic lymph nodes. The pathology from brain metastasis was reported as "metastatic choriocarcinoma" (a germ cell tumor) by local pathologists. She initiated carboplatin and etoposide, a regimen for choriocarcinoma. Subsequently, her case was assessed by pathologists from an academic cancer center, who gave the diagnosis of "adenocarcinoma with aberrant expression of ß-hCG" and finally pathologists at our hospital, who gave the diagnosis of "poorly differentiated carcinoma with choriocarcinoma features". Genomic profiling detected a KRAS G13R mutation and transcriptomics profiling was suggestive of lung origin. The patient was treated with carboplatin/paclitaxel/ipilimumab/nivolumab followed by consolidation radiation therapy. She had no evidence of progression to date, 16 months after the initial presentation. The molecular profiling could facilitate diagnosing of challenging cancer cases. In addition, chemoimmunotherapy and local consolidation radiation therapy may provide promising therapeutic options for patients with lung cancer exhibiting choriocarcinoma features.

Structural and molecular bases of rod photoreceptor morphogenesis and disease.

The rod cell has an extraordinarily specialized structure that allows it to carry out its unique function of detecting individual photons of light. Both the structural features of the rod and the metabolic processes required for highly amplified light detection seem to have rendered the rod especially sensitive to structural and metabolic defects, so that a large number of gene defects are primarily associated with rod cell death and give rise to blinding retinal dystrophies. The structures of the rod, especially those of the sensory cilium known as the outer segment, have been the subject of structural, biochemical, and genetic analysis for many years, but the molecular bases for rod morphogenesis and for cell death in rod dystrophies are still poorly understood. Recent developments in imaging technology, such as cryo-electron tomography and super-resolution fluorescence microscopy, in gene sequencing technology, and in gene editing technology are rapidly leading to new breakthroughs in our understanding of these questions. A summary is presented of our current understanding of selected aspects of these questions, highlighting areas of uncertainty and contention as well as recent discoveries that provide new insights. Examples of structural data from emerging imaging technologies are presented.

Three-dimensional architecture of murine rod cilium revealed by cryo-EM.

The connecting cilium of the rod photoreceptor is a tubular structure that bridges two adjacent cellular compartments, the inner segment, the major site of biosynthesis and energy metabolism, and the outer segment, a highly specialized ciliary structure responsible for phototransduction. The connecting cilium allows for active processes of protein sorting and transport to occur between them. Mutations affecting the cargo, their transporters, and the structural components of the primary cilium and basal body lead to aberrant trafficking and photoreceptor cell death. Understanding the overall design of the cilium, its architectural organization, and the function of varied protein complexes within the structural hierarchy of the cilium requires techniques for visualizing their native three-dimensional structures at high magnification. Here we describe methods for isolating retinas from mice, purifying fragments of rod cells that include much of the inner segment and the rod photoreceptor cilia, vitrifying the cell fragments, and determining their structures by cryo-electron tomography.

Integration of multiscale dendritic spine structure and function data into systems biology models.

Comprising 10(11) neurons with 10(14) synaptic connections the human brain is the ultimate systems biology puzzle. An increasing body of evidence highlights the observation that changes in brain function, both normal and pathological, consistently correlate with dynamic changes in neuronal anatomy. Anatomical changes occur on a full range of scales from the trafficking of individual proteins, to alterations in synaptic morphology both individually and on a systems level, to reductions in long distance connectivity and brain volume. The major sites of contact for synapsing neurons are dendritic spines, which provide an excellent metric for the number and strength of signaling connections between elements of functional neuronal circuits. A comprehensive model of anatomical changes and their functional consequences would be a holy grail for the field of systems neuroscience but its realization appears far on the horizon. Various imaging technologies have advanced to allow for multi-scale visualization of brain plasticity and pathology, but computational analysis of the big data sets involved forms the bottleneck toward the creation of multiscale models of brain structure and function. While a full accounting of techniques and progress toward a comprehensive model of brain anatomy and function is beyond the scope of this or any other single paper, this review serves to highlight the opportunities for analysis of neuronal spine anatomy and function provided by new imaging technologies and the high-throughput application of older technologies while surveying the strengths and weaknesses of currently available computational analytical tools and room for future improvement.

TRP channel gene expression in the mouse retina.

In order to identify candidate cation channels important for retinal physiology, 28 TRP channel genes were surveyed for expression in the mouse retina. Transcripts for all TRP channels were detected by RT-PCR and sequencing. Northern blotting revealed that mRNAs for 12 TRP channel genes are enriched in the retina. The strongest signals were observed for TRPC1, TRPC3, TRPM1, TRPM3, and TRPML1, and clear signals were obtained for TRPC4, TRPM7, TRPP2, TRPV2, and TRPV4. In situ hybridization and immunofluorescence revealed widespread expression throughout multiple retinal layers for TRPC1, TRPC3, TRPC4, TRPML1, PKD1, and TRPP2. Striking localization of enhanced mRNA expression was observed for TRPC1 in the photoreceptor inner segment layer, for TRPM1 in the inner nuclear layer (INL), for TRPM3 in the INL, and for TRPML1 in the outer plexiform and nuclear layers. Strong immunofluorescence signal in cone outer segments was observed for TRPM7 and TRPP2. TRPC5 immunostaining was largely confined to INL cells immediately adjacent to the inner plexiform layer. TRPV2 antibodies stained photoreceptor axons in the outer plexiform layer. Expression of TRPM1 splice variants was strong in the ciliary body, whereas TRPM3 was strongly expressed in the retinal pigmented epithelium.