Protein complexes associated with cellular processes comprise a significant fraction of all biology, but our understanding of their heterogeneous organization remains inadequate, particularly for physiological densities of multiple protein species. Recent advances in microscopy allow us to localize within cells both individual proteins and some of the interactions between proteins predicted by immunoprecipitation and genetic complementation, but the sheer number of protein species involved in any one biological structure is increasingly becoming a limiting factor. Many-color immuno-histochemistry, while flexible and selective, requires primary antibodies that are either directly coupled to fluorophores or from many diverse species, often forcing a shift to suboptimally affine primary antibodies. Although available immuno-histochemical labels can be supplemented with expressed markers and other methods, their potential permutations still present an overwhelming hurdle for current microscopy methods. Alternatives, such as the expression of tags for subsequent labeling, positively impact the rate at which different molecules can be imaged, but protein colocalization remains speculative with these techniques. Additionally, techniques exist to enable antibody reuse in unembedded tissue with a variety of elution methods, but the precision of subsequent labeling and tissue damage has not been documented. Towards resolving this limitation, this year we report a new technique based on resin-embedded multicycle imaging (REMI) of proteins in-situ. By stabilizing protein structure and antigenicity in acrylic resins, affinity labels were repeatedly applied, imaged, removed, and replaced. In principle, an arbitrarily large number of proteins of interest may be imaged on the same specimen with subsequent digital overlay. A series of novel preparative methods were developed to address the problem of imaging multiple protein species in areas of the plasma membrane or volumes of cytoplasm of individual cells. For multiplexed examination of antibody staining we used straightforward computational techniques to align sequential images, and super-resolution microscopy was used to further define membrane protein colocalization. We give one example of a fibroblast membrane with eight multiplexed proteins. A simple statistical analysis of this limited membrane proteomic dataset is sufficient to demonstrate the analytical power contributed by additional imaged proteins when studying membrane protein domains. Recessive osteogenesis imperfecta (OI) is caused by defects in proteins involved in post-translational interactions with type I collagen. Recently, a novel form of moderately severe OI caused by null mutations in TMEM38B was identified. TMEM38B encodes the ER membrane monovalent cation channel, TRIC-B, proposed to counterbalance IP3R-mediated Ca2+ release from intracellular stores. The molecular mechanisms by which TMEM38B mutations cause OI are unknown. We identified 3 probands with recessive defects in TMEM38B. TRIC-B protein is undetectable in proband fibroblasts and osteoblasts, although reduced TMEM38B transcripts are present. TRIC-B deficiency causes impaired release of ER luminal Ca2+, associated with deficient store-operated calcium entry, although SERCA and IP3R have normal stability. To determine whether the decreased Ca2+ flux in TRIC-B deficient cells was associated with abnormal Ca2+ steady state levels within the ER, we measured free ER Ca2+ using the targeted ratiometric calcium sensor D1ER. Interestingly, there was no measurable change in ER luminal free Ca2+ in proband or normal control cells following ATP treatment, suggesting that the amount of stored Ca2+ needed to elevate cytoplasmic Ca2+ is minor compared to total Ca2+ available in the ER. Furthermore, no significant differences in the baseline (before ATP treatment) and depleted (after ionomycin treatment) ER Ca2+ signals were observed between normal control and proband cells (Fig 3E). This indicates that TRIC-B deficiency does not result in an altered steady state luminal ER free Ca2+ as, for example, a result of the abnormal Ca2+ flux. Luminal ER free Ca2+ is neither decreased nor increased. The disturbed Ca2+ flux causes ER stress and increased BiP, and dysregulates synthesis of proband type I collagen at multiple steps. Collagen helical lysine hydroxylation is reduced, while telopeptide hydroxylation is increased, despite increased LH1 and decreased Ca2+-dependent FKBP65, respectively. Although PDI levels are maintained, procollagen chain assembly is delayed in proband cells. The resulting misfolded collagen is substantially retained in TRIC-B null cells, consistent with a 50-70% reduction in secreted collagen. Lower-stability forms of collagen that elude proteasomal degradation are not incorporated into extracellular matrix, which contains only normal stability collagen, resulting in matrix insufficiency. These data support a role for TRIC-B in intracellular Ca2+ homeostasis, and demonstrate that absence of TMEM38B causes OI by dysregulation of calcium flux kinetics in the ER, impacting multiple collagen-specific chaperones and modifying enzymes.
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