The mitochondrial inner membrane can reshape under different physiological conditions. How, at which frequency this occurs in living cells, and the molecular players involved are unknown. Here, we show using state‐of‐the‐art live‐cell stimulated emission depletion (STED) super‐resolution nanoscopy that neighbouring crista junctions (CJs) dynamically appose and separate from each other in a reversible and balanced manner in human cells. Staining of cristae membranes (CM), using various protein markers or two lipophilic inner membrane‐specific dyes, further revealed that cristae undergo continuous cycles of membrane remodelling. These events are accompanied by fluctuations of the membrane potential within distinct cristae over time. Both CJ and CM dynamics depended on MIC13 and occurred at similar timescales in the range of seconds. Our data further suggest that MIC60 acts as a docking platform promoting CJ and contact site formation. Overall, by employing advanced imaging techniques including fluorescence recovery after photobleaching (FRAP), single‐particle tracking (SPT), live‐cell STED and high‐resolution Airyscan microscopy, we propose a model of CJ dynamics being mechanistically linked to CM remodelling representing cristae membrane fission and fusion events occurring within individual mitochondria.

bSarcolemmal membrane-associated protein (SLMAP) is a tail-anchored protein involved in fundamental cellular processes, such as myoblast fusion, cell cycle progression, and chromosomal inheritance. Further, SLMAP misexpression is associated with endothelial dysfunctions in diabetes and cancer. SLMAP is part of the conserved striatin-interacting phosphatase and kinase (STRIPAK) complex required for specific signaling pathways in yeasts, filamentous fungi, insects, and mammals. In filamentous fungi, STRIPAK was initially discovered in Sordaria macrospora, a model system for fungal differentiation. Here, we functionally characterize the STRIPAK subunit PRO45, a homolog of human SLMAP. We show that PRO45 is required for sexual propagation and cell-to-cell fusion and that its forkhead-associated (FHA) domain is essential for these processes. Protein-protein interaction studies revealed that PRO45 binds to STRIPAK subunits PRO11 and SmMOB3, which are also required for sexual propagation. Superresolution structured-illumination microscopy (SIM) further established that PRO45 localizes to the nuclear envelope, endoplasmic reticulum, and mitochondria. SIM also showed that localization to the nuclear envelope requires STRIPAK subunits PRO11 and PRO22, whereas for mitochondria it does not. Taken together, our study provides important insights into fundamental roles of the fungal SLMAP homolog PRO45 and suggests STRIPAK-related and STRIPAK-unrelated functions. Membrane recruitment of protein complexes, cell signaling modules, and enzymes is a critical step for many cellular functions. The family of tail-anchored proteins is recognized for anchoring proteins and vesicles to specific membranes, such as the endoplasmic reticulum (ER) and the outer mitochondrial membrane (1), and tail-anchored proteins are characterized by a C-terminal single transmembrane domain, which is posttranslationally inserted into membranes (2, 3).Sarcolemmal membrane-associated protein (SLMAP) is a tailanchored protein first identified in myocardiac cells (4). In mammals, this protein is known to be involved in myoblast fusion during embryonic development, excitation-contraction coupling in cardiac myocytes, and cell cycle progression (5-8). Furthermore, SLMAP was identified to be a disease gene for Brugada syndrome, a cardiac channelopathy (9). The functional diversity of SLMAP is dependent on alternative splicing, leading to at least four different isoforms of the protein (4, 6, 7, 10). Importantly, gene expression analyses have implicated SLMAP misexpression with endothelial dysfunctions in diabetes, chromosomal aberrations, and cancer (11-14), and currently, SLMAP is the target of lectin-based treatment of drug-resistant cancer cells (15).SLMAP is conserved from yeasts to humans, and characterized fungal SLMAP homologs include Neurospora crassa HAM-4 (hyphal anastomosis 4), Saccharomyces cerevisiae Far9p (factor arrest 9p) and Far10p, as well as Schizosaccharomyces pombe Csc1p (component of SIP complex 1p) (16-18). HAM-4 is essential for vegetativ...

SummaryDuring Drosophila embryogenesis, the first epithelium with defined cortical compartments is established during cellularization. Actin polymerization is required for the separation of lateral and basal domains as well as suppression of tubular extensions in the basal domain. The actin nucleator mediating this function is unknown. We found that the formin Diaphanous (Dia) is required for establishing and maintaining distinct lateral and basal domains during cellularization. In dia mutant embryos lateral marker proteins, such as Discs-large and Armadillo/β-Catenin spread into the basal compartment. Furthermore, high-resolution and live-imaging analysis of dia mutant embryos revealed an increased number of membrane extensions and endocytic activity at the basal domain, indicating a suppressing function of dia on membrane invaginations. Dia function might be based on an antagonistic interaction with the F-BAR protein Cip4/Toca-1, a known activator of the WASP/WAVE-Arp2/3 pathway. Dia and Cip4 physically and functionally interact and overexpression of Cip4 phenocopies dia loss-of-function. In vitro, Cip4 inhibits mainly actin nucleation by Dia. Thus, our data support a model in which linear actin filaments induced by Dia stabilize cortical compartmentalization by antagonizing membrane turnover induced by WASP/WAVE-Arp2/3.

Ciliopathies are life‐threatening human diseases caused by defective cilia. They can often be traced back to mutations of genes encoding transition zone (TZ) proteins demonstrating that the understanding of TZ organisation is of paramount importance. The TZ consists of multimeric protein modules that are subject to a stringent assembly hierarchy. Previous reports place Rpgrip1l at the top of the TZ assembly hierarchy in Caenorhabditis elegans. By performing quantitative immunofluorescence studies in RPGRIP1L−/− mouse embryos and human embryonic cells, we recognise a different situation in vertebrates in which Rpgrip1l deficiency affects TZ assembly in a cell type‐specific manner. In cell types in which the loss of Rpgrip1l alone does not affect all modules, additional truncation or removal of vertebrate‐specific Rpgrip1 results in an impairment of all modules. Consequently, Rpgrip1l and Rpgrip1 synergistically ensure the TZ composition in several vertebrate cell types, revealing a higher complexity of TZ assembly in vertebrates than in invertebrates.

Post-transcriptional regulation of gene expression occurs by multiple mechanisms, including subcellular localization of mRNA and alteration of the poly(A) tail length. These mechanisms play crucial roles in the dynamics of cell polarization and embryonic development. Furthermore, mRNAs are emerging therapeutics and chemical alterations to increase their translational efficiency are highly sought after. We show that yeast poly(A) polymerase can be used to install multiple azido-modified adenosine nucleotides to luciferase and eGFP-mRNAs. These mRNAs can be efficiently reacted in a bioorthogonal click reaction with fluorescent reporters without degradation and without sequence alterations in their coding or untranslated regions. Importantly, the modifications in the poly(A) tail impact positively on the translational efficiency of reporter-mRNAs in vitro and in cells. Therefore, covalent fluorescent labeling at the poly(A) tail presents a new way to increase the amount of reporter protein from exogenous mRNA and to label genetically unaltered and translationally active mRNAs.

Edited by Velia M. Fowler Whereas myosin 18B (Myo18B) is known to be a critical sarcomeric protein, the function of myosin 18A (Myo18A) is unclear, although it has been implicated in cell motility and Golgi shape. Here, we show that homozygous deletion (homozygous tm1a, tm1b, or tm1d alleles) of Myo18a in mouse is embryonic lethal. Reminiscent of Myo18b, Myo18a was highly expressed in the embryo heart, and cardiac-restricted Myo18a deletion in mice was embryonic lethal. Surprisingly, using Western blot analysis, we were unable to detect the known isoforms of Myo18A, Myo18A␣ and Myo18A␤, in mouse heart using a custom C-terminal antibody. However, alternative anti-Myo18A antibodies detected a larger than expected protein, and RNA-Seq analysis indicated that a novel Myo18A transcript is expressed in mouse ventricular myocytes (and human heart). Cloning and sequencing revealed that this cardiac isoform, denoted Myo18A␥, lacks the PDZ-containing N terminus of Myo18A␣ but includes an alternative N-terminal extension and a long serine-rich C terminus. EGFP-tagged Myo18A␥ expressed in ventricular myocytes localized to the level of A-bands in sarcomeres, and Myo18a knockout embryos at day 10.5 exhibited disorganized sarcomeres with wavy thick filaments. We additionally generated myeloid-restricted Myo18a knockout mice to investigate the role of Myo18A in nonmuscle cells, exemplified by macrophages, which express more Myo18A␤ than Myo18A␣, but no defects in cell shape, motility, or Golgi shape were detected. In summary, we have identified a previously unrecognized sarcomere component, a large novel isoform (denoted Myo18A␥) of Myo18A. Thus, both members of class XVIII myosins are critical components of cardiac sarcomeres.Myosins are a superfamily of motor proteins, encoded in mice by 39 genes, which have been categorized into 12 classes (I, II (conventional myosins), III, V, VI, VII, IX, X, XV, XVI, XVIII, and XIX) (1, 2). Class XVIII myosins consist of Myo18A and Myo18B. The longest known splice variant of Myo18A (denoted Myo18A␣), initially termed MysPDZ (myosin containing PDZ domain) (3), has a distinctive N-terminal extension, which includes a highly charged KE (lysine-glutamine)rich region and a PDZ domain (3-5). In contrast, a shorter splice variant of Myo18A (denoted Myo18A␤) lacks the N-terminal extension (4, 5). Like Myo18A␣, Myo18B (as distinct from Myo18A␤) has a long N-terminal extension but lacks a PDZ domain (6). Myo18B was shown to localize to the Z-discs of striated muscle, and Myo18b deficiency causes embryonic lethality associated with disordered sarcomeres (7). In contrast, Berger et al. (8) found that Myo18B-GFP localizes to sarcomeric A-bands, rather than Z-discs, in zebrafish skeletal muscle, and gene deletion impaired sarcomere assembly. Thus, Myo18B is thought to be important for the development and/or maintenance of sarcomeres.The physiological function of Myo18A, which is widely expressed (3), is not clear. In 2003, Mori et al. (4) inferred that MysPDZ␣-YFP 2 (Myo18A␣-YFP) localizes to the Golgi...

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There was an error published in J. Cell Sci. 128, 499-515.On p. 507 of this article, the legend of Fig. 6 contained an incomplete description of panel B.The correct sentence should read:(B) Wild type stage 4 egg chamber stained for Armadillo and E-cadherin; arrowhead indicates apical E-cadherin localization in follicle cells.We apologise to the readers for any confusion that this error might have caused.