An isoprenylation and palmitoylation motif promotes intraluminal vesicle delivery of proteins in cells from distant species

12 p.-7 fig.The C-terminal ends of small GTPases contain hypervariable sequences which may be posttranslationally modified by defined lipid moieties. The diverse structural motifs generated direct proteins towards specific cellular membranes or organelles. However, knowledge on the factors that determine these selective associations is limited. Here we show, using advanced microscopy, that the isoprenylation and palmitoylation motif of human RhoB (–CINCCKVL) targets chimeric proteins to intraluminal vesicles of endolysosomes in human cells, displaying preferential co-localization with components of the late endocytic pathway. Moreover, this distribution is conserved in distant species, including cells from amphibians, insects and fungi. Blocking lipidic modifications results in accumulation of CINCCKVL chimeras in the cytosol, from where they can reach endolysosomes upon release of this block. Remarkably, CINCCKVL constructs are sorted to intraluminal vesicles in a cholesterol-dependent process. In the lower species, neither the C-terminal sequence of RhoB, nor the endosomal distribution of its homologs are conserved; in spite of this, CINCCKVL constructs also reach endolysosomes in Xenopus laevis and insect cells. Strikingly, this behavior is prominent in the filamentous ascomycete fungus Aspergillus nidulans, in which GFP-CINCCKVL is sorted into endosomes and vacuoles in a lipidation-dependent manner and allows monitoring endosomal movement in live fungi. In summary, the isoprenylated and palmitoylated CINCCKVL sequence constitutes a specific structure which delineates an endolysosomal sorting strategy operative in phylogenetically diverse organisms.This work was supported by grants SAF2009-11642 and SAF2012-36519 from MINECO and RETIC RD12/0013/0008 to DPS, grants BIO2012-30695 fromcMINECO and S2010/BMD-2414 from Comunidad de Madrid to MAP, and grants from the Norwegian Cancer Society and the South-Eastern Norway Regional Health Authority to HS. CLO is supported by the FPI program from MINECO (BES-2010-033718). The stay of CLO at HS laboratory was supported by the short stay grant EEBB-I-12-04482 from MINECO.Peer reviewe

A CEP104-CSPP1 Complex Is Required for Formation of Primary Cilia Competent in Hedgehog Signaling

CEP104 is an evolutionarily conserved centrosomal and ciliary tip protein. CEP104 loss-of-function mutations are reported in patients with Joubert syndrome, but their function in the etiology of ciliopathies is poorly understood. Here, we show that cep104 silencing in zebrafish causes cilia-related manifestations: shortened cilia in Kupffer's vesicle, heart laterality, and cranial nerve development defects. We show that another Joubert syndrome-associated cilia tip protein, CSPP1, interacts with CEP104 at microtubules for the regulation of axoneme length. We demonstrate in human telomerase reverse transcriptase-immortalized retinal pigmented epithelium (hTERT-RPE1) cells that ciliary translocation of Smoothened in response to Hedgehog pathway stimulation is both CEP104 and CSPP1 dependent. However, CEP104 is not required for the ciliary recruitment of CSPP1, indicating that an intra-ciliary CEP104-CSPP1 complex controls axoneme length and Hedgehog signaling competence. Our in vivo and in vitro analyses of CEP104 define its interaction with CSPP1 as a requirement for the formation of Hedgehog signaling-competent cilia, defects that underlie Joubert syndrome

3D-Structured Illumination Microscopy of Centrosomes in Human Cell Lines

The centrosome is the main microtubule-organizing center of animal cells, and is composed of two barrel-shaped microtubule-based centrioles embedded in protein dense pericentriolar material. Compositional and architectural re-organization of the centrosome drives its duplication, and enables its microtubule-organizing activity and capability to form the primary cilium, which extends from the mature (mother) centriole, as the cell exits the cell cycle. Centrosomes and primary cilia are essential to human health, signified by the causal role of centrosome- and cilia-aberrations in numerous congenic disorders, as well as in the etiology and progression of cancer. The list of disease-associated centrosomal proteins and their proximitomes is steadily expanding, emphasizing the need for high resolution mapping of such proteins to specific substructures of the organelle. Here, we provide a detailed 3D-structured illumination microscopy (3D-SIM) protocol for comparative localization analysis of fluorescently labeled proteins at the centrosome in fixed human cell lines, at approximately 120 nm lateral and 300 nm axial resolution. The procedure was optimized to work with primary antibodies previously known to depend on more disruptive fixation reagents, yet largely preserves centriole and centrosome architecture, as shown by transposing acquired images of landmark proteins on previously published transmission electron microscopy (TEM) images of centrosomes. Even more advantageously, it is compatible with fluorescent protein tags. Finally, we introduce an internal reference to ensure correct 3D channel alignment. This protocol hence enables flexible, swift, and information-rich localization and interdependence analyses of centrosomal proteins, as well as their disorder-associated mutations

Ribosomal readthrough at a short UGA stop codon context triggers dual localization of metabolic enzymes in fungi and animals

Translation of mRNA into a polypeptide chain is a highly accurate process. Many prokaryotic and eukaryotic viruses, however, use leaky termination of translation to optimize their coding capacity. Although growing evidence indicates the occurrence of ribosomal readthrough also in higher organisms, a biological function for the resulting extended proteins has been elucidated only in very few cases. Here, we report that in human cells programmed stop codon readthrough is used to generate peroxisomal isoforms of cytosolic enzymes. We could show for NAD-dependent lactate dehydrogenase B (LDHB) and NAD-dependent malate dehydrogenase 1 (MDH1) that translational readthrough results in C-terminally extended protein variants containing a peroxisomal targeting signal 1 (PTS1). Efficient readthrough occurs at a short sequence motif consisting of a UGA termination codon followed by the dinucleotide CU. Leaky termination at this stop codon context was observed in fungi and mammals. Comparative genome analysis allowed us to identify further readthrough-derived peroxisomal isoforms of metabolic enzymes in diverse model organisms. Overall, our study highlights that a defined stop codon context can trigger efficient ribosomal readthrough to generate dually targeted protein isoforms. We speculate that beyond peroxisomal targeting stop codon readthrough may have also other important biological functions, which remain to be elucidated

CSPP-L Associates with the Desmosome of Polarized Epithelial Cells and Is Required for Normal Spheroid Formation

<div><p>Deleterious mutations of the Centrosome/Spindle Pole associated Protein 1 gene, <i>CSPP1</i>, are causative for Joubert-syndrome and Joubert-related developmental disorders. These disorders are defined by a characteristic mal-development of the brain, but frequently involve renal and hepatic cyst formation. CSPP-L, the large protein isoform of <i>CSPP1</i> localizes to microtubule ends of the mitotic mid-spindle and the ciliary axoneme, and is required for ciliogenesis. We here report the microtubule independent but Desmoplakin dependent localization of CSPP-L to Desmosomes in apical-basal polarized epithelial cells. Importantly, siRNA conferred depletion of CSPP-L or Desmoplakin promoted multi-lumen spheroid formation in 3D-cultures of non-ciliated human colon carcinoma Caco-2 cells. Multi-lumen spheroids of <i>CSPP1</i> siRNA transfectants showed disrupted apical cell junction localization of the cytoskeleton organizing RhoGEF ECT2. Our results hence identify a novel, non-ciliary role for CSPP-L in epithelial morphogenesis.</p></div

Nanoscopy on the Chea(i)p

Super-resolution microscopy allows for stunning images with a resolution well beyond the optical diffraction limit, but the imaging techniques are demanding in terms of instrumentation and software. Using scientific-grade cameras, solid-state lasers and top-shelf microscopy objective lenses drives the price and complexity of the system, limiting its use to well-funded institutions. However, by harnessing recent developments in CMOS image sensor technology and low-cost illumination strategies, super-resolution microscopy can be made available to the mass-markets for a fraction of the price. Here, we present a 3D printed, self-contained super-resolution microscope with a price tag below 1000 $ including the objective and a cellphone. The system relies on a cellphone to both acquire and process images as well as control the hardware, and a photonic-chip enabled illumination. The system exhibits 100nm optical resolution using single-molecule localization microscopy and can provide live super-resolution imaging using light intensity fluctuation methods. Furthermore, due to its compactness, we demonstrate its potential use inside bench-top incubators and high biological safety level environments imaging SARS-CoV-2 viroids. By the development of low-cost instrumentation and by sharing the designs and manuals, the stage for democratizing super-resolution imaging is set.B.S.A. acknowledges funding from European Commission ERC Starting Grant (336716) and ERC PoC 957464 and ), Research Council of Norway (Grant 288565). We acknowledge funding by the DFG Transregio Project TRR166, TP04 (PT) and the Leibniz ScienceCampus InfectoOptics SAS-2015-HKI-LWC (AJ). The authors further acknowledge the support of this work by a grant from the IZKF (ACSP02) (SDE). The study was further funded by the Deutsche Forschungsge-meinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy (EXC 2051, Project-ID 390713860). This work was financially supported by the Deutsche Forschungsge- 10 meinschaft through the Cluster of Excellence “Balance of the Microverse” under Germany’s Excellence Strategy – EXC 2051 – Project-ID 690 390713860. We thank Fatina Siwczak and Swen Carlstedt for fruitful discussions. We further thank Merete Storflor and S¨oren Abel (Infection Biology Research Group, UiT) for providing the Ecoli samples and Deanna Wolfson (Optics group, UiT) for help with preparation of the Ecoli samples. We would like to thank Sindy Burgold-Voigt and Ralf Ehricht from Leibniz IPHT Jena for their great methodical contribution and the fruitful discussions. We thank especially Ingo Fuchs who helped a lot in understanding the principles of the acquisition process of cellphone cameras as well as his support of the software design. Pablo Carravilla acknowledges the Basque Government postdoctoral program (POS 2019 2 0022) for funding his position. pMDLg/pRRE and pRSV-Rev were a gift from Didier Trono (Addgene plasmid #12251 and #12253; http://n2t.net/addgene:12251 and http://n2t.net/addgene:12253; RRID:Addgene 12251 and RRID:Addgene 12253). K.O.S is supported by a Career fellowship from the South-Eastern Norway Regional Health AuthorityN

Depletion of CSPP-L and Desmoplakin cause multi-lumen spheroid formation in Caco-2 spheroids.

<p>(A) Localization of CSPP-L (a-CSPP-L, green), filamentous actin (Phalloidin, white), E-cadherin (a-E-cadherin, red), and DNA (blue) during different stages of spheroid development of Caco-2 cells. Cells were grown in 3D-Matrigel culture and formalin fixed for IF. Images show projections of z-sections enclosing the entire lumen volume. CSPP-L shows prominent enrichment juxtapose to the apical filamentous actin throughout all stages of spheroid development (B) The apical CSPP-L staining pattern (a-CSPP-L, green) is CSPP1 siRNA sensitive and not altered by Desmoplakin depletion (a-α-tubulin, red; phalloidin, white). CSPP1 and Desmoplakin siRNA Caco-2 transfectants develop disorganized cell aggregates with multiple lumen.</p

Desmoplakin is required for CSPP-L localization to the Desmosome.

<p>IF (A-C) and immunoblotting of total cell lysates (D) of HCC1937 cells treated with indicated siRNAs. Cells were transfected in low calcium medium. 72hrs post-transfection cells were allowed to form cell-cell junctions for 40 min by change to pre-warmed, normal calcium medium. Cell-cell junction staining of CSPP-L (green in overlay image) is siCSPP1 sensitive (A). Depletion of CSPP-L does not impair cell-cell junction localization of Desmoplakin (A, red) or β-catenin (B, red). Depletion of Desmoplakin (C, red) results in loss of CSPP-L (green) staining at cell-cell junctions. Knockdown efficacy was monitored by immunoblotting for Desmoplakin, CSPP-L and compared to the loading control γ-tubulin (D).</p

Apical-basal polarity is not disrupted in CSPP-L and Desmoplakin depleted multi-lumen Caco-2 spheroids.

<p>(A) Quantification of the multi-lumen phenotype in Caco-2 siRNA transfectants (siGFP, siCSPP1, siCSPP1 <i>s</i>mart <i>p</i>ool, siDSP). The bar diagram shows average of two experiments, error bars depict standard deviation. Statistical significance was tested by paired t-test. (B) CSPP-L and Desmoplakin depletion in Caco-2 spheroids was validated by immunoblotting (right panel). (C) Multi-lumen spheroids in CSPP1 and Desmoplakin depleted cells depict filamentous actin stabilization indicated by solid arrow heads (Phalloidin, white) and PKCζ enrichment (a-PKCζ, red) at the lumen facing apical membrane. Occasional weak PKCζ staining at the basal-side of outer-rim cells is seen in <i>siCSPP1</i> and <i>siDSP</i> transfectants (open arrowheads). (D) Centrosomes (a-Pericentrin, green) positioned in the lumen oriented cytoplasm (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134789#pone.0134789.s003" target="_blank">S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134789#pone.0134789.s004" target="_blank">S2</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134789#pone.0134789.s005" target="_blank">S3</a> Videos).</p