The coordinated integration of ribosomal RNA and protein into two functional ribosomal subunits is safeguarded by quality control checkpoints that ensure ribosomes are correctly assembled and functional before they engage in translation. Quality control is critical in maintaining the integrity of ribosomes and necessary to support healthy cell growth and prevent diseases associated with mistakes in ribosome assembly. Its importance is demonstrated by the finding that bypassing quality control leads to misassembled, malfunctioning ribosomes with altered translation fidelity, which change gene expression and disrupt protein homeostasis. In this review, we outline our understanding of quality control within ribosome synthesis and how failure to enforce quality control contributes to human disease. We first provide a definition of quality control to guide our investigation, briefly present the main assembly steps, and then examine stages of assembly that test ribosome function, establish a pass-fail system to evaluate these functions, and contribute to altered ribosome performance when bypassed, and are thus considered "quality control."

Proper chromosome segregation is crucial for cell division. In eukaryotes, this is achieved by the kinetochore, an evolutionarily conserved multiprotein complex that physically links the DNA to spindle microtubules and takes an active role in monitoring and correcting erroneous spindle-chromosome attachments. Our mechanistic understanding of these functions and how they ensure an error-free outcome of mitosis is still limited, partly because we lack a complete understanding of the kinetochore structure in the cell. In this study, we use single-molecule localization microscopy to visualize individual kinetochore complexes in situ in budding yeast. For major kinetochore proteins, we measured their abundance and position within the metaphase kinetochore. Based on this comprehensive dataset, we propose a quantitative model of the budding yeast kinetochore. While confirming many aspects of previous reports based on bulk imaging, our results present a unifying nanoscale model of the kinetochore in budding yeast.

Acute Promyelocytic Leukemia is caused by expression of the oncogenic Promyelocytic Leukemia (PML)-Retinoic Acid Receptor Alpha (RARA) fusion protein. Therapy with arsenic trioxide results in degradation of PML-RARA and PML and cures the disease. Modification of PML and PML-RARA with SUMO and ubiquitin precedes ubiquitin-mediated proteolysis. To identify additional components of this pathway, we performed proteomics on PML bodies. This revealed that association of p97/VCP segregase with PML bodies is increased after arsenic treatment. Pharmacological inhibition of p97 altered the number, morphology, and size of PML bodies, accumulated SUMO and ubiquitin modified PML and blocked arsenic-induced degradation of PML-RARA and PML. p97 localized to PML bodies in response to arsenic, and siRNA-mediated depletion showed that p97 cofactors UFD1 and NPLOC4 were critical for PML degradation. Thus, the UFD1-NPLOC4-p97 segregase complex is required to extract poly-ubiquitinated, poly-SUMOylated PML from PML bodies, prior to degradation by the proteasome.

ER tubules form and maintain membrane contact sites (MCSs) with late endosomes/lysosomes (LE/lys). The molecular composition and cellular functions of these MCSs are poorly understood. Here, we find that Tex2, an SMP domain-containing lipid transfer protein conserved in metazoan and yeast, is a tubular ER protein and is recruited to ER-LE/lys MCSs by TMEM55, phosphatases that convert PI(4,5)P2 to PI5P on LE/lys. We show that the Tex2-TMEM55 interaction occurs between an N-terminal region of Tex2 and a catalytic motif in the PTase domain of TMEM55. The Tex2-TMEM55 interaction can be regulated by endosome-resident type 2 PI4K activities. Functionally, Tex2 knockout results in defects in lysosomal trafficking, digestive capacity, and lipid composition of LE/lys membranes. Together, our data identify Tex2 as a tubular ER protein that resides at TMEM55-dependent ER-LE/lys MCSs required for lysosomal functions.

Along myelinated axons, Shaker-type potassium channels (Kv1) accumulate at high density in the juxtaparanodal region, directly adjacent to the paranodal axon-glia junctions that flank the nodes of Ranvier. However, the mechanisms that control the clustering of Kv1 channels, as well as their function at this site, are still poorly understood. Here we demonstrate that axonal ADAM23 is essential for both the accumulation and stability of juxtaparanodal Kv1 complexes. The function of ADAM23 is critically dependent on its interaction with its extracellular ligands LGI2 and LGI3. Furthermore, we demonstrate that juxtaparanodal Kv1 complexes affect the refractory period, thus enabling high-frequency burst firing of action potentials. Our findings not only reveal a previously unknown molecular pathway that regulates Kv1 channel clustering, but they also demonstrate that the juxtaparanodal Kv1 channels that are concealed below the myelin sheath, play a significant role in modifying axonal physiology.

Hepatocytes grow their apical surfaces anisotropically to generate a 3D network of bile canaliculi (BC). BC elongation is ensured by apical bulkheads, membrane extensions that traverse the lumen and connect juxtaposed hepatocytes. We hypothesize that apical bulkheads are mechanical elements that shape the BC lumen in liver development but also counteract elevated biliary pressure. Here, by resolving their structure using STED microscopy, we found that they are sealed by tight junction loops, connected by adherens junctions, and contain contractile actomyosin, characteristics of mechanical function. Apical bulkheads persist at high pressure upon microinjection of fluid into the BC lumen, and laser ablation demonstrated that they are under tension. A mechanical model based on ablation results revealed that apical bulkheads double the pressure BC can hold. Apical bulkhead frequency anticorrelates with BC connectivity during mouse liver development, consistent with predicted changes in biliary pressure. Our findings demonstrate that apical bulkheads are load-bearing mechanical elements that could protect the BC network against elevated pressure.

The essential COPI coat mediates retrieval of transmembrane proteins at the Golgi and endosomes following recruitment by the small GTPase, Arf1. ArfGAP proteins regulate COPI coats, but molecular details for COPI recognition by ArfGAPs remain elusive. Biochemical and biophysical data reveal how β'-COP propeller domains directly engage the yeast ArfGAP, Glo3, with a low micromolar binding affinity. Calorimetry data demonstrate that both β'-COP propeller domains are required to bind Glo3. An acidic patch on β'-COP (D437/D450) interacts with Glo3 lysine residues located within the BoCCS (binding of coatomer, cargo, and SNAREs) region. Targeted point mutations in either Glo3 BoCCS or β'-COP abrogate the interaction in vitro, and loss of the β'-COP/Glo3 interaction drives Ste2 missorting to the vacuole and aberrant Golgi morphology in budding yeast. These data suggest that cells require the β'-COP/Glo3 interaction for cargo recycling via endosomes and the TGN, where β'-COP serves as a molecular platform to coordinate binding to multiple proteins, including Glo3, Arf1, and the COPI F-subcomplex.

Septins are filamentous GTPases that play important but poorly characterized roles in ciliogenesis. Here, we show that SEPTIN9 regulates RhoA signaling at the base of cilia by binding and activating the RhoA guanine nucleotide exchange factor, ARHGEF18. GTP-RhoA is known to activate the membrane targeting exocyst complex, and suppression of SEPTIN9 causes disruption of ciliogenesis and mislocalization of an exocyst subunit, SEC8. Using basal body-targeted proteins, we show that upregulating RhoA signaling at the cilium can rescue ciliary defects and mislocalization of SEC8 caused by global SEPTIN9 depletion. Moreover, we demonstrate that the transition zone components, RPGRIP1L and TCTN2, fail to accumulate at the transition zone in cells lacking SEPTIN9 or depleted of the exocyst complex. Thus, SEPTIN9 regulates the recruitment of transition zone proteins on Golgi-derived vesicles by activating the exocyst via RhoA to allow the formation of primary cilia.

Microtubules are dynamic cytoskeletal polymers, and their organization and stability are tightly regulated by numerous cellular factors. While regulatory proteins controlling the formation of interphase microtubule arrays and mitotic spindles have been extensively studied, the biochemical mechanisms responsible for generating stable microtubule cores of centrioles and cilia are poorly understood. Here, we used in vitro reconstitution assays to investigate microtubule-stabilizing properties of CSPP1, a centrosome and cilia-associated protein mutated in the neurodevelopmental ciliopathy Joubert syndrome. We found that CSPP1 preferentially binds to polymerizing microtubule ends that grow slowly or undergo growth perturbations and, in this way, resembles microtubule-stabilizing compounds such as taxanes. Fluorescence microscopy and cryo-electron tomography showed that CSPP1 is deposited in the microtubule lumen and inhibits microtubule growth and shortening through two separate domains. CSPP1 also specifically recognizes and stabilizes damaged microtubule lattices. These data help to explain how CSPP1 regulates the elongation and stability of ciliary axonemes and other microtubule-based structures.

The widespread use of fluorescence microscopy has prompted the ongoing development of tools aiming to improve resolution and quantification accuracy for study of biological questions. Current calibration and quantification tools for fluorescence images face issues with usability/user experience, lack of automation, and comprehensive multidimensional measurement/correction capabilities. Here, we developed 3D-Speckler, a versatile, and high-throughput image analysis software that can provide fluorescent puncta quantification measurements such as 2D/3D particle size, spatial location/orientation, and intensities through semi-automation in a single, user-friendly interface. Integrated analysis options such as 2D/3D local background correction, chromatic aberration correction, and particle matching/filtering are also encompassed for improved precision and accuracy. We demonstrate 3D-Speckler microscope calibration capabilities by determining the chromatic aberrations, field illumination uniformity, and response to nanometer-scale emitters above and below the diffraction limit of our imaging system using multispectral beads. Furthermore, we demonstrated 3D-Speckler quantitative capabilities for offering insight into protein architectures and composition in cells.

Caveolae are small membrane invaginations that generally are stably attached to the plasma membrane. Their release is believed to depend on the GTPase dynamin 2 (Dyn2), in analogy with its role in fission of clathrin-coated vesicles. The mechanistic understanding of caveola fission is, however, sparse. Here, we used microscopy-based tracking of individual caveolae in living cells to determine the role of Dyn2 in caveola dynamics. We report that Dyn2 stably associated with the bulb of a subset of caveolae, but was not required for formation or fission of caveolae. Dyn2-positive caveolae displayed longer plasma membrane duration times, whereas depletion of Dyn2 resulted in shorter duration times and increased caveola fission. The stabilizing role of Dyn2 was independent of its GTPase activity and the caveola stabilizing protein EHD2. Thus, we propose that, in contrast to the current view, Dyn2 is not a core component of the caveolae machinery, but rather functions as an accessory protein that restrains caveola internalization.

The key to ensuring proper chromosome segregation during mitosis is the kinetochore (KT), a tightly regulated multiprotein complex that links the centromeric chromatin to the spindle microtubules and as such leads the segregation process. Understanding its architecture, function, and regulation is therefore essential. However, due to its complexity and dynamics, only its individual subcomplexes could be studied in structural detail so far. In this study, we construct a nanometer-precise in situ map of the human-like regional KT of Schizosaccharomyces pombe using multi-color single-molecule localization microscopy. We measure each protein of interest (POI) in conjunction with two references, cnp1CENP-A at the centromere and sad1 at the spindle pole. This allows us to determine cell cycle and mitotic plane, and to visualize individual centromere regions separately. We determine protein distances within the complex using Bayesian inference, establish the stoichiometry of each POI and, consequently, build an in situ KT model with unprecedented precision, providing new insights into the architecture.

The transcription factor Prdm16 functions as a potent suppressor of transforming growth factor-beta (TGF-β) signaling, whose inactivation is deemed essential to the progression of pancreatic ductal adenocarcinoma (PDAC). Using the KrasG12D-based mouse model of human PDAC, we surprisingly found that ablating Prdm16 did not block but instead accelerated PDAC formation and progression, suggesting that Prdm16 might function as a tumor suppressor in this malignancy. Subsequent genetic experiments showed that ablating Prdm16 along with Smad4 resulted in a shift from a well-differentiated and confined neoplasm to a highly aggressive and metastatic disease, which was associated with a striking deviation in the trajectory of the premalignant lesions. Mechanistically, we found that Smad4 interacted with and recruited Prdm16 to repress its own expression, therefore pinpointing a model in which Prdm16 functions downstream of Smad4 to constrain the PDAC malignant phenotype. Collectively, these findings unveil an unprecedented antagonistic interaction between the tumor suppressors Smad4 and Prdm16 that functions to restrict PDAC progression and metastasis.

The primary method for probing DNA replication dynamics is DNA fiber analysis, which utilizes thymidine analog incorporation into nascent DNA, followed by immunofluorescent microscopy of DNA fibers. Besides being time-consuming and prone to experimenter bias, it is not suitable for studying DNA replication dynamics in mitochondria or bacteria, nor is it adaptable for higher-throughput analysis. Here, we present mass spectrometry-based analysis of nascent DNA (MS-BAND) as a rapid, unbiased, quantitative alternative to DNA fiber analysis. In this method, incorporation of thymidine analogs is quantified from DNA using triple quadrupole tandem mass spectrometry. MS-BAND accurately detects DNA replication alterations in both the nucleus and mitochondria of human cells, as well as bacteria. The high-throughput capability of MS-BAND captured replication alterations in an E. coli DNA damage-inducing gene library. Therefore, MS-BAND may serve as an alternative to the DNA fiber technique, with potential for high-throughput analysis of replication dynamics in diverse model systems.

The dynamin-like GTPase atlastin is believed to be the minimal machinery required for homotypic endoplasmic reticulum (ER) membrane fusion, mainly because Drosophila atlastin is sufficient to drive liposome fusion. However, it remains unclear whether mammalian atlastins, including the three human atlastins, are sufficient to induce liposome fusion, raising doubts about their major roles in mammalian cells. Here, we show that all human atlastins are sufficient to induce fusion when reconstituted into liposomes with a lipid composition mimicking that of the ER. Although the fusogenic activity of ATL1, which is predominantly expressed in neuronal cells, was weaker than that of ATL2 or ATL3, the addition of M1-spastin, a neuron-specific factor, markedly increased ATL1-mediated liposome fusion. Although we observed efficient fusion between ER microsomes isolated from cultured, non-neuronal cells that predominantly express ATL2-1, an autoinhibited isoform of ATL2, ATL2-1 failed to support liposome fusion by itself as reported previously, indicating that cellular factors enable ATL2-1 to mediate ER fusion in vivo.

Cellular cryo-electron tomography (cryo-ET) enables three-dimensional reconstructions of organelles in their native cellular environment at subnanometer resolution. However, quantifying ultrastructural features of pleomorphic organelles in three dimensions is challenging, as is defining the significance of observed changes induced by specific cellular perturbations. To address this challenge, we established a semiautomated workflow to segment organellar membranes and reconstruct their underlying surface geometry in cryo-ET. To complement this workflow, we developed an open-source suite of ultrastructural quantifications, integrated into a single pipeline called the surface morphometrics pipeline. This pipeline enables rapid modeling of complex membrane structures and allows detailed mapping of inter- and intramembrane spacing, curvedness, and orientation onto reconstructed membrane meshes, highlighting subtle organellar features that are challenging to detect in three dimensions and allowing for statistical comparison across many organelles. To demonstrate the advantages of this approach, we combine cryo-ET with cryo-fluorescence microscopy to correlate bulk mitochondrial network morphology (i.e., elongated versus fragmented) with membrane ultrastructure of individual mitochondria in the presence and absence of endoplasmic reticulum (ER) stress. Using our pipeline, we demonstrate ER stress promotes adaptive remodeling of ultrastructural features of mitochondria including spacing between the inner and outer membranes, local curvedness of the inner membrane, and spacing between mitochondrial cristae. We show that differences in membrane ultrastructure correlate to mitochondrial network morphologies, suggesting that these two remodeling events are coupled. Our pipeline offers opportunities for quantifying changes in membrane ultrastructure on a single-cell level using cryo-ET, opening new opportunities to define changes in ultrastructural features induced by diverse types of cellular perturbations.