Universal sample preparation method for proteome analysis

Microscopy and mass spectrometry (MS) are complementary techniques: The former provides spatiotemporal information in living cells, but only for a handful of recombinant proteins at a time, whereas the latter can detect thousands of endogenous proteins simultaneously, but only in lysed samples. Here, we introduce technology that combines these strengths by offering spatially and temporally resolved proteomic maps of endogenous proteins within living cells. Our method relies on a genetically targetable peroxidase enzyme that biotinylates nearby proteins, which are subsequently purified and identified by MS. We used this approach to identify 495 proteins within the human mitochondrial matrix, including 31 not previously linked to mitochondria. The labeling was exceptionally specific and distinguished between inner membrane proteins facing the matrix versus the intermembrane space (IMS). Several proteins previously thought to reside in the IMS or outer membrane, including protoporphyrinogen oxidase, were reassigned to the matrix by our proteomic data and confirmed by electron microscopy. The specificity of peroxidase-mediated proteomic mapping in live cells, combined with its ease of use, offers biologists a powerful tool for understanding the molecular composition of living cells.

Proteomic Mapping of Mitochondria in Living Cells via Spatially Restricted Enzymatic Tagging

During division, eukaryotic cells undergo a dramatic, complex and coordinated remodelling of their cytoskeleton and membranes. For cell division to occur, chromosomes must be segregated and new cellular structures, such as the spindle apparatus, must be assembled. Pre-existing organelles, such as the nuclear envelope, endoplasmic reticulum and Golgi apparatus, must be disassembled or remodelled, distributed and reformed. Smaller organelles such as mitochondria as well as cytoplasmic content must also be properly distributed between daughter cells. This mixture of organelles and cytoplasm is bound by a plasma membrane that is itself subject to remodelling as division progresses. The lipids resident in these different membrane compartments play important roles in facilitating the division process. In recent years, we have begun to understand how membrane remodelling is coordinated during division; however, there is still much to learn. In this Review, we discuss recent insights into how these important cellular events are performed and regulated. During cell division, the distribution of membrane-bound organelles needs to be tightly regulated to ensure the proper composition and function of daughter cells. Recent studies have shed light on the range of complex and dynamic mechanisms needed to mediate organelle inheritance and membrane remodelling during cell division.

Membrane and organelle dynamics during cell division

Cellular Reorganization during Mitotic Entry

Cells depend on hugely diverse lipidomes for many functions. The actions and structural integrity of the plasma membrane and most organelles also critically depend on membranes and their lipid comp...

Lipid Cell Biology: A Focus on Lipids in Cell Division

Cell division requires a coordinated action of the cell cycle machinery, cytoskeletal elements, chromosomes, and membranes. Cell division studies have greatly benefitted from the mass spectrometry (MS)-based proteomic approaches for probing the biochemistry of highly dynamic complexes and their coordination with each other as a cell progresses into division. In this review, the authors first summarize a wide-range of proteomic studies that focus on the identification of sub-cellular components/protein complexes of the cell division machinery including kinetochores, mitotic spindle, midzone, and centrosomes. The authors also highlight MS-based large-scale analyses of the cellular components that are largely understudied during cell division such as the cell surface and lipids. Then, the authors focus on posttranslational modification analyses, especially phosphorylation and the resulting crosstalk with other modifications as a cell undergoes cell division. Combining proteomic approaches that probe the biochemistry of cell division components with functional genomic assays will lead to breakthroughs toward a systems-level understanding of cell division.

Proteomics in Cell Division.

Cell migration requires spatiotemporally coordinated activities of multicomponent structures including the actomyosin cortex, plasma membrane, adhesion complexes and the polarity proteins. How they function together to drive this complex dynamic process remains an outstanding question. Here, we show that a member of the protocadherin family, PCDH7 displays a polarized localization in migratory cells with a dynamic enrichment at the leading and rear edges. Perturbation of PCDH7 interferes with the migration of nontransformed retinal pigment epithelial cells and invasion of cancer cells. The overexpression of PCDH7 enhances the migration capability of cortical neurons in vivo. PCDH7 interacts with the myosin phosphatase subunits MYPT1 and PP1cβ and it enhances the phosphorylation of regulatory light chain and ERM at the leading and rear edges of migratory cells. The chemical inhibition of phosphatase activity recovers migration phenotypes of PCDH7 knockout cells. We propose that PCDH7 regulate phosphorylation thus activity of myosin and ERM at the polarized cortex by quenching myosin phosphatase that results in a higher persistence of migrating cells. Collectively, our study suggests a new mechanism for the spatial coordination of plasma membrane and the cortex during cell migration.

https://www.biorxiv.org/content/biorxiv/early/2021/09/21/2021.09.21.460794.full.pdf

PCDH7 Promotes Cell Migration by Regulating Myosin Activity

Adaptive tracking algorithm for trajectory analysis of cells and layer-by-layer assessment of motility dynamics

Type A aortic dissection (AD) is a devastating cardiovascular emergency requiring emergent surgical intervention. Most patients with AD have several risk factors for the disease including longstanding hypertension, smoking history, atherosclerosis, and old age. Younger patients may also present with AD if a genetic disorder affecting the integrity of the aorta is present. This case presents an otherwise healthy 36-year-old male with no known significant family history who presented with an atypical presentation of aortic dissection. He described a five-day history of chest pressure made worse with exertion followed by progressive dyspnea which prompted him to seek medical attention. His initial laboratory workup revealed an elevated troponin I level which prompted a cardiology consultation in the emergency department. Transthoracic echocardiography revealed dilatation of the aortic root and aortic regurgitation. CT angiography of the chest was performed revealing a type A dissection beginning at the aortic root and terminating proximal to the right brachiocephalic artery. Involvement of the coronary arteries was suspected due to the elevated troponin I. He was taken to the operating room and underwent aortic grafting, right coronary artery bypass, and repair of the left main artery. Unfortunately, at the end of the operation, the patient went into refractory ventricular fibrillation, which progressed to asystole. He was unable to be revived.

/pdf/type-a-aortic-dissection-presenting-as-acute-coronary-2jlgrh3j.pdf

Type A Aortic Dissection Presenting as Acute Coronary Syndrome in a Young Male Patient: A Case Report

Successful cell division requires dramatic reorganization of the cell cortex in coordination with actomyosin cytoskeleton organization, membrane trafficking and cell adhesion. Although the contractile actomyosin ring is considered as hallmark of cytokinesis, in some cell types cell adhesion systems have been shown to drive cytokinesis independently from actomyosin function. We previously reported that Protocadherin 7 (PCDH7) localizes to the mitotic cortex which is required for building up the full mitotic rounding pressure. Here, we show that PCDH7 localizes to the mitotic cell cortex and to the cleavage furrow by a palmitoylation-dependent mechanism. At the cleavage furrow, PCDH7 facilitates the activation of myosin II and successful cytokinesis. Strikingly, PCDH7 promotes cytokinesis even when the myosin II contractility and integrin mediated adhesion are blocked. This work describes a palmitoylation-dependent cortical reorganization which promotes cytokinesis under different conditions.

https://www.biorxiv.org/content/biorxiv/early/2020/05/24/2020.05.24.111831.full.pdf

Protocadherin 7 localizes to the plasma membrane during mitosis and promotes cytokinesis by a palmitoylation-dependent mechanism

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