Object detection networks and augmented reality for cellular detection in fluorescence microscopy

Object detection networks are high-performance algorithms famously applied to the task of identifying and localizing objects in photography images. We demonstrate their application for the classification and localization of cells in fluorescence microscopy by benchmarking four leading object detection algorithms across multiple challenging 2D microscopy datasets. Furthermore we develop and demonstrate an algorithm that can localize and image cells in 3D, in close to real time, at the microscope using widely available and inexpensive hardware. Furthermore, we exploit the fast processing of these networks and develop a simple and effective augmented reality (AR) system for fluorescence microscopy systems using a display screen and back-projection onto the eyepiece. We show that it is possible to achieve very high classification accuracy using datasets with as few as 26 images present. Using our approach, it is possible for relatively nonskilled users to automate detection of cell classes with a variety of appearances and enable new avenues for automation of fluorescence microscopy acquisition pipelines. © 2020 Waithe et al

Fluidity and lipid composition of membranes of peroxisomes, mitochondria and the ER from oleic acid-induced saccharomyces cerevisiae

The maintenance of a fluid lipid bilayer is key for organelle function and cell viability. Given the critical role of lipid compositions in determining membrane properties and organelle identity, it is clear that cells must have elaborate mechanism for membrane maintenance during adaptive responses to environmental conditions. Emphasis of the presented study is on peroxisomes, oleic acid-inducible organelles that are essential for the growth of yeast under conditions of oleic acid as single carbon source. Here, we isolated peroxisomes, mitochondria and ER from oleic acid-induced Saccharomyces cerevisiae and determined the lipid composition of their membranes using shotgun lipidomics and compared it to lipid ordering using fluorescence microscopy. In comparison to mitochondrial and ER membranes, the peroxisomal membranes were slightly more disordered and characterized by a distinct enrichment of phosphaditylinositol, indicating an important role of this phospholipid in peroxisomal membrane associated processes

Intracellular Photophysics of an Osmium Complex bearing an Oligothiophene Extended Ligand

This contribution describes the excited-state properties of an Osmium-complex when taken up into human cells. The complex 1 [Os(bpy)2(IP-4T)](PF6)2 with bpy=2,2′-bipyridine and IP-4T=2-{5′-[3′,4′-diethyl-(2,2′-bithien-5-yl)]-3,4-diethyl-2,2′-bithiophene}imidazo[4,5-f][1,10]phenanthroline) can be discussed as a candidate for photodynamic therapy in the biological red/NIR window. The complex is taken up by MCF7 cells and localizes rather homogeneously within in the cytoplasm. To detail the sub-ns photophysics of 1, comparative transient absorption measurements were carried out in different solvents to derive a model of the photoinduced processes. Key to rationalize the excited-state relaxation is a long-lived 3ILCT state associated with the oligothiophene chain. This model was then tested with the complex internalized into MCF7 cells, since the intracellular environment has long been suspected to take big influence on the excited state properties. In our study of 1 in cells, we were able to show that, though the overall model remained the same, the excited-state dynamics are affected strongly by the intracellular environment. Our study represents the first in depth correlation towards ex-vivo and in vivo ultrafast spectroscopy for a possible photodrug. © 2020 The Authors. Published by Wiley-VCH Gmb

Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5

Cellular functions rely on proper actions of organelles such as peroxisomes. These organelles rely on the import of proteins from the cytosol. The peroxisomal import receptor PEX5 takes up target proteins in the cytosol and transports them to the peroxisomal matrix. However, its cytosolic molecular interactions have so far not directly been disclosed. Here, we combined advanced optical microscopy and spectroscopy techniques such as fluorescence correlation spectroscopy and stimulated emission depletion microscopy with biochemical tools to present a detailed characterization of the cytosolic diffusion and interaction dynamics of PEX5. Among other features, we highlight a slow diffusion of PEX5, independent of aggregation or target binding, but associated with cytosolic interaction partners via its N-terminal domain. This sheds new light on the functionality of the receptor in the cytosol as well as highlighting the potential of using complementary microscopy tools to decipher molecular interactions in the cytosol by studying their diffusion dynamics.We acknowledge funding by the Wolfson Foundation, MRC (grant no. MC_UU_12010/unit programs G0902418 and MC_UU_12025), the Wellcome Trust (grant no. 104924/14/Z/14, Strategic Award 091911 (Micron)), MRC/BBSRC/EPSRC (grant no. MR/K01577X/1, MRC grant no. MC_UU_12010/unit programs G0902418 and MC_UU_12025), the EPA Cephalosporin Fund, the John Fell Fund, and the Deutsche Forschungsgemeinschaft (research unit 1905 “Structure and function of the peroxisomal translocon”; grant no. 322325142 “Super-resolution optical microscopy studies of peroxisomal protein import in the yeast Saccharomyces cerevisiae”, Germany′s Excellence Strategy – EXC 2051 – Project-ID 390713860, project number 316213987 – SFB 1278). P. C. acknowledges a postdoctoral fellowship from the Basque Government (POS_2018_1_0066 and POS_2019_2_0022).Peer reviewe

Bedeutung und Mechanismen des peroxisomalen Matrix‐Protein‐Importes des deubiquitinierenden Enzyms USP2

USP2 liegt in vier Isoformen vor, die verschiedenste Funktionen, insbesondere bei der Regulation der Apoptose, innehaben. Eine Überexpression der drei kurzen Isoformen 2, 3 und 4 führte zu einer verstärkten Caspase-Aktivität. Alle vier Isoformen des USP2 werden PEX5 abhängig über ein PTS1-Signal in die peroxisomale Matrix transportiert. Dieser Import ist aufgrund einer geringen Affinität zu PEX5 nicht sehr effizient, so dass ein großer Teil des USP2 im Zytosol verbleibt. Durch Mutation des PTS1 von USP2-3 konnte die Affinität zum Rezeptor und somit die peroxisomale Importeffizienz erhöht werden. Daraufhin zeigte sich eine Verringerung der Caspase-Aktivität, woraus sich schließen lässt, dass USP2-3 nur im Zytosol seine pro-apoptotische Funktion ausführen kann. Dies legt nahe, dass die eigentliche Funktion des peroxisomalen Imports von USP2-3 einen Regulationsmechanismus der Apoptose darstellt, indem es von seinen Interaktionspartnern in Zytosol und Zellkern räumlich getrennt wird

Unexpectedly High Levels of Inverted Re-Insertions Using Paired sgRNAs for Genomic Deletions

Use of dual sgRNAs is a common CRISPR/Cas9-based strategy for the creation of genetic deletions. The ease of screening combined with a rather high rate of success makes this approach a reliable genome engineering procedure. Recently, a number of studies using CRISPR/Cas9 have revealed unwanted large-scale rearrangements, duplications, inversions or larger-than-expected deletions. Strict quality control measures are required to validate the model system, and this crucially depends on knowing which potential experimental outcomes to expect. Using the dual sgRNA deletion approach, our team discovered high levels of excision, inversion and re-insertion at the site of targeting. We detected those at a variety of genomic loci and in several immortalized cell lines, demonstrating that inverted re-insertions are a common by-product with an overall frequency between 3% and 20%. Our findings imply an inherent danger in the misinterpretation of screening data when using only a single PCR screening. While amplification of the region of interest might classify clones as wild type (WT) based on amplicon size, secondary analyses can discover heterozygous (HET) clones among presumptive WTs, and events deemed as HET clones could potentially be full KO. As such, screening for inverted re-insertions helps in decreasing the number of clones required to obtain a full KO. With this technical note, we want to raise awareness of this phenomenon and suggest implementing a standard secondary PCR while screening for deletions

Ligand-Induced Compaction of the PEX5 Receptor-Binding Cavity Impacts Protein Import Efficiency into Peroxisomes

Peroxisomes entirely rely on the import of their proteome across the peroxisomal membrane. Recognition efficiencies of peroxisomal proteins vary by more than 1000-fold, but the molecular rationale behind their subsequent differential import and sorting has remained enigmatic. Using the protein cargo alanine-glyoxylate aminotransferase as a model, an unexpected increase from 34 to 80% in peroxisomal import efficiency of a single-residue mutant has been discovered. By high-resolution structural analysis, we found that it is the recognition receptor PEX5 that adapts its conformation for high-affinity binding rather than the cargo protein signal motif as previously thought. During receptor recognition, the binding cavity of the receptor shrinks to one third of its original volume. This process is impeded in the wild-type protein cargo because of a bulky side chain within the recognition motif, which blocks contraction of the PEX5 binding cavity. Our data provide a new insight into direct protein import efficiency by removal rather than by addition of an apparent specific sequence signature that is generally applicable to peroxisomal matrix proteins and to other receptor recognition processes

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The proapoptotic effect of USP2-3 overexpression is reduced by optimization of the peroxisomal import.

<p><b>A</b>: Activity of caspase 3/7 in HEK-293 cells after expression of different USP2-3 variants. Cells were transfected with the empty control plasmid pcDNA3.1 or plasmids, encoding the indicated USP2-3 variants. 24 h after transfection caspase activity was measured by a luminescence assay. Expression of USP2-3 did result in an increase of the caspase activity, while caspase acitivity upon expression of the inactive USP2-3 C34A variant was only slightly above the control. The apoptotic effect of the different USP2-3 variants differ significantly, which was shown by one way ANOVA analysis (p<0.0001). However, in comparison to the unmodified protein, the caspase activity was significantly decreased upon expression USP2-3 SRM>SKL, which was shown by a Tukey’s multiple comparisons test (*p<0.05). Mean values are shown with standard deviation from three independent experiments, which were performed in triplicate for each USP2 variant. <b>B</b>: Activity of Caspase 3/7 of FlpUSP2-3 cells after transfection with the empty vector or plasmids coding for PEX5 or mutated PEX5 C11A, which does not support peroxisomal protein import. 48 h after transfection, caspase activity was monitored by a luminescence assay (upper panel). The caspase activity significantly differs upon the expressed PEX5 variants (one way ANOVA analysis; p<0.001). Tukey’s multiple comparisons test revealed that in comparison to the control (empty vector), transfection with the import defective PEX5 C11A mutant had no significant effect on the caspase activity, but was significantly decreased after transfection of wild-type PEX5 (***p<0.005). Shown are mean values with standard deviation from three independent experiments, which were performed in triplicate for each construct. Expression of the different PEX5 variants was analyzed by immunoblotting (lower panel). Prohibitin was used as a loading control. PEX5, USP2-3 and Prohibitin were detected with specific antibodies. (RUL = <i>Relative Light Unit</i>).</p

Peroxisomal Import Reduces the Proapoptotic Activity of Deubiquitinating Enzyme USP2

<div><p>The human deubiquitinating enzyme ubiquitin-specific protease 2 (USP2) regulates multiple cellular pathways, including cell proliferation and apoptosis. As a result of alternative splicing four USP2 isoenzymes are expressed in human cells of which all contain a weak peroxisome targeting signal of type 1 (PTS1) at their C-termini. Here, we systematically analyzed apoptotic effects induced by overexpression and intracellular localization for each isoform. All isoforms exhibit proapoptotic activity and are post-translationally imported into the matrix of peroxisomes in a PEX5-dependent manner. However, a significant fraction of the USP2 pool resides in the cytosol due to a weaker PTS1 and thus low affinity to the PTS receptor PEX5. Blocking of peroxisomal import did not interfere with the proapoptotic activity of USP2, suggesting that the enzyme performs its critical function outside of this compartment. Instead, increase of the efficiency of USP2 import into peroxisomes either by optimization of its peroxisomal targeting signal or by overexpression of the PTS1 receptor did result in a reduction of the apoptotic rate of transfected cells. Our studies suggest that peroxisomal import of USP2 provides additional control over the proapoptotic activity of cytosolic USP2 by spatial separation of the deubiquitinating enzymes from their interaction partners in the cytosol and nucleus.</p></div