Score: 92.3, Published: 2024-02-11
DOI: 10.1101/2024.02.11.579797
While protein homeostasis is a hallmark of gene regulation, unraveling the hidden regulatory mechanisms that maintain homeostasis is difficult using traditional methods. To confront this problem, we CRISPR engineered a human cell line with multiple tags in the endogenous MYH9 gene, which encodes the essential and ubiquitous myosin-2A cytoskeletal motor. Using these cells, we imaged MYH9 transcription, translation, and mature mRNA and protein in distinct colors, enabling a full dissection of the central dogma. Our data show that MYH9 transcription is upregulated in an SRF-dependent manner in response to cytoskeletal cues and that MYH9 translation can either buffer or match the transcriptional response depending on context. Upon knockdown of actin-depolymerizing proteins like cofilin, translation efficiency drops by a factor of two to buffer strong transcriptional upregulation, likely to help prevent excessive myosin activity. In contrast, following serum stimulation, translation matches the transcriptional response to readily establish equilibrium. Our results identify contextual translational buffering as an important regulatory mechanism driving stable MYH9 expression. They also demonstrate the power and broad applicability of our cell line, which can now be used to accurately quantify central dogma dynamics in response to diverse forms of cellular perturbations.
Score: 18.6, Published: 2024-02-16
DOI: 10.1101/2024.02.16.580695
The definitive demonstration of protein localization on primary cilia has been a challenge for cilia biologists. Primary cilia are solitary thread-like projections that contain specialized protein composition, but as the ciliary structure overlays the cell membrane and other cell parts, the identity of ciliary proteins are difficult to ascertain by conventional imaging approaches like immunofluorescence microscopy. Surface scanning electron microscopy combined with immuno-labeling (immuno-SEM) bypasses some of these indeterminacies by unambiguously showing protein expression in the context of the 3D ultrastructure of the cilium. Here we apply immuno-SEM to specifically identify proteins on the primary cilia of mouse and human pancreatic islets, including post-translationally modified tubulin, intraflagellar transport (IFT) 88, the small GTPase Arl13b, as well as subunits of axonemal dynein. Key parameters in sample preparation, immuno-labeling, and imaging acquisition are discussed to facilitate similar studies by others in the cilia research community.
Score: 17.6, Published: 2024-02-14
DOI: 10.1101/2024.02.14.580304
Ciliopathies are often caused by defects in the ciliary microtubule core. Glutamylation is abundant in cilia, and its dysregulation may contribute to ciliopathies and neurodegeneration. Mutation of the deglutamylase CCP1 causes infantile-onset neurodegeneration. In C. elegans, ccpp-1 loss causes age-related ciliary degradation that is suppressed by mutation in the conserved NEK10 homolog nekl-4. NEKL-4 is absent from cilia, yet negatively regulates ciliary stability via an unknown, glutamylation-independent mechanism. We show that NEKL-4 was mitochondria-associated. nekl-4 mutants had longer mitochondria, a higher baseline mitochondrial oxidation state, and suppressed ccpp-1 mutant lifespan extension in response to oxidative stress. A kinase-dead nekl-4(KD) mutant ectopically localized to ccpp-1 cilia and rescued degenerating microtubule doublet B-tubules. A nondegradable nekl-4(PEST{Delta}) mutant resembled the ccpp-1 mutant with dye filling defects and B-tubule breaks. The nekl-4(PEST{Delta}) Dyf phenotype was suppressed by mutation in the depolymerizing kinesin-8 KLP-13/KIF19A. We conclude that NEKL-4 influences ciliary stability by activating ciliary kinesins and promoting mitochondrial homeostasis. SummaryNeurodegeneration and ciliary degeneration are caused by mutation of the deglutamylase CCP1/CCPP-1 in humans and C. elegans. The conserved NIMA-related kinase NEKL-4/NEK10 can suppress or promote degeneration in an activity-dependent manner that involves cilia-mitochondria communication and that is independent of glutamylation.
Score: 17.4, Published: 2024-02-14
DOI: 10.1101/2024.02.13.580110
Toxoplasma gondii is a protozoan parasite that has evolved a developmental morphotype called tachyzoite that navigates between cells and moves in and out of them in a wide repertoire of homeothermic hosts. Relying on a uniquely constant apicobasal bipolarity coupled to an actomyosin-driven retrograde surface flow, the tachyzoite has elaborated a molecular machinery to assemble transient anchoring contacts with the environment, which support the traction force required to power a typical helical gliding motility. Combining micropatterning with live, reflection interference contrast and expansion microscopies, we bring first nanoscale evidence that the tachyzoite needs to build only one apical anchoring contact with the substrate, thus spatially defining a minimal force transmission platform over which it can slide. We uncover that the apicobasal driven surface flow is set up in response to extracellular biochemical cues independent of adhesin release and tachyzoite-surface interactions, hence prior to motile activity. Furthermore, to identify the minimal adhesion requirements for helical gliding at the level of individual molecular species, we combine biochemical and biophysical quantitative assays based on tunable surface chemistry and quartz crystal microbalance with dissipation monitoring. These approaches uncover that glycosaminoglycan (GAG)-parasite interactions are sufficient to promote a productive contact for helical gliding and pave the way for the characterization of the structure and density of the molecules functionally engaged at this essential parasite-substrate mechanosensitive interface.
Score: 14.6, Published: 2024-02-15
DOI: 10.1101/2024.02.14.580336
Autophagic mechanisms that maintain nuclear envelope homeostasis are bulwarks to aging and disease. By leveraging 4D lattice light sheet microscopy and correlative light and electron tomography, we define a quantitative and ultrastructural timeline of a nuclear macroautophagy (nucleophagy) pathway in yeast. Nucleophagy initiates with a rapid local accumulation of the nuclear cargo adaptor Atg39 at the nuclear envelope adjacent to the nucleus-vacuole junction and is delivered to the vacuole in [~]300 seconds through an autophagosome intermediate. Mechanistically, nucleophagy incorporates two consecutive and genetically defined membrane fission steps: inner nuclear membrane (INM) fission generates a lumenal vesicle in the perinuclear space followed by outer nuclear membrane (ONM) fission to liberate a double membraned vesicle to the cytosol. ONM fission occurs independently of phagophore engagement and instead relies surprisingly on dynamin-like protein1 (Dnm1), which is recruited to sites of Atg39 accumulation at the nuclear envelope. Loss of Dnm1 compromises nucleophagic flux by stalling nucleophagy after INM fission. Our findings reveal how nuclear and INM cargo are removed from an intact nucleus without compromising its integrity, achieved in part by a non-canonical role for Dnm1 in nuclear envelope remodeling.
Score: 13.7, Published: 2024-02-08
DOI: 10.1101/2024.02.06.579089
Rhodamines have been continuously optimized in brightness, biocompatibility, and colors to fulfill the demands of modern bioimaging. However, the problem of phototoxicity caused by the excited fluorophore under long-term illumination has been largely neglected, hampering their use in time-lapse imaging. Here we introduce cyclooctatetraene (COT) conjugated rhodamines that span the visible spectrum and exhibit significantly reduced phototoxicity. We identified a general strategy for the generation of Gentle Rhodamines, which preserved their outstanding spectroscopic properties and cell permeability while showing an efficient reduction of singlet-oxygen formation and diminished cellular photodamage. Paradoxically, their photobleaching kinetics do not go hand in hand with reduced phototoxicity. By combining COT-conjugated spirocyclization motifs with targeting moieties, these gentle rhodamines compose a toolkit for time-lapse imaging of mitochondria, DNA, and actin and synergize with covalent and exchangeable HaloTag labeling of cellular proteins with less photodamage than their commonly used precursors. Taken together, the Gentle Rhodamines generally offer alleviated phototoxicity and allow advanced video recording applications, including voltage imaging.
Score: 12.8, Published: 2024-02-14
DOI: 10.1101/2024.02.13.580097
Centrosomes are membrane-less organelles that orchestrate a wide array of biological functions by acting as microtubule organizing centers. Recently, the centrosome has been implicated in caspase-1 activation and inflammasome-driven pyroptosis. Here, we report that caspase-2-driven apoptosis is elicited in blood cells that fail cytokinesis and that extra centrosomes are necessary to trigger this cell death. Activation of caspase-2 depends on the PIDDosome multi-protein complex and priming of PIDD1 at extra centrosomes is critical for this signalling pathway. Accordingly, loss of its centrosomal adapter, ANKRD26, allows for cell survival and unrestricted polyploidization in response to cytokinesis failure. Mechanistically, cell death is initiated upstream of mitochondria and caspase-9 via caspase-2-mediated processing of the proapoptotic BCL2 family protein BID, driving BAX/BAK-dependent mitochondrial outer membrane permeabilization (MOMP). Remarkably, BID-deficient cells enforce apoptosis by engaging a p53-dependent pro-apoptotic transcriptional response initiated by caspase-2. Consistently, MDM2 and BID act as shared caspase-2 substrates that synergize to promote cell killing. Our findings document that the centrosome limits its own unscheduled duplication by the induction of PIDDosome-driven mitochondrial apoptosis to avoid potentially pathogenic polyploidization events.
Score: 11.3, Published: 2024-02-16
DOI: 10.1101/2024.02.16.580263
Mitochondrial DNA is exposed to multiple insults produced by normal cellular function. Upon mtDNA replication stress the mitochondrial genome transfers to endosomes where it is degraded. Here, using proximity proteomics we found that mtDNA replication stress leads to the rewiring of the mitochondrial proximity proteome, increasing mitochondria association with lysosomal and vesicle-associated proteins, such as the GTPase RAB10 and the retromer. We found that upon mtDNA replication stress, RAB10 enhances mitochondrial fragmentation and relocates from the ER to lysosomes containing mtDNA. The retromer enhances and coordinates the expulsion of mitochondrial matrix components through mitochondrial-derived vesicles, and mtDNA with direct transfer to lysosomes. Using a Drosophila model carrying a long deletion on the mtDNA (deltamtDNA), we evaluated in vivo the role of the retromer in mtDNA extraction and turnover in the larval epidermis. The presence of deltamtDNA elicits the activation of a specific transcriptome profile related to counteract mitochondrial damage. Expression of the retromer component Vps35 is sufficient to restore mtDNA homoplasmy and mitochondrial defects associated with deltamtDNA. Our data reveal novel regulators involved in the specific elimination of mtDNA. We demonstrate that modulation of the retromer in vivo is a successful mechanism to restore mitochondrial function associated with mtDNA damage.
Score: 11.0, Published: 2024-02-15
DOI: 10.1101/2024.02.14.579514
Prohibitins are a highly conserved family of proteins that have been implicated in a variety of functions including mitochondrial stress signalling and housekeeping, cell cycle progression, apoptosis, lifespan regulation and many others1, 2. The human prohibitins PHB1 and PHB2 have been proposed to act as scaffolds within the mitochondrial inner membrane, but their molecular organisation remained elusive. Using an integrative structural biology approach combining quantitative Western blotting, cryo-electron tomography, subtomogram averaging and molecular modelling, we determined the molecular organisation of the human prohibitin complex within the mitochondrial inner membrane. The proposed bell-shaped structure consists of eleven alternating PHB1 and PHB2 molecules. This study reveals an average of about 43 prohibitin complexes per crista, covering 1-3 % of the cristae membranes. These findings provide a structural basis for understanding the functional contributions of prohibitins to the integrity and spatial organisation of the mitochondrial inner membrane.
Score: 8.9, Published: 2024-02-15
DOI: 10.1101/2024.02.15.580237
Tissue maintenance is underpinned by resident stem cells, whose activity is modulated by microenvironmental cues. Using Drosophila as a simple model to identify regulators of stem cell behaviour and survival in vivo, we have identified novel connections between the conserved transmembrane proteoglycan Syndecan, nuclear properties and stem cell function. In the Drosophila midgut, Syndecan depletion in intestinal stem cells results in their loss from the tissue, impairing tissue renewal. At the cellular level, Syndecan depletion alters cell and nuclear shape, and causes nuclear lamina invaginations and DNA damage. In a second tissue, the developing Drosophila brain, live imaging revealed that Syndecan depletion in neural stem cells results in nuclear envelope remodelling defects which arise upon cell division. Our findings reveal a new role for Syndecan in the maintenance of nuclear properties in diverse stem cell types.