DeepMIB : User-friendly and open-source software for training of deep learning network for biological image segmentation

We present DeepMIB, a new software package that is capable of training convolutional neural networks for segmentation of multidimensional microscopy datasets on any workstation. We demonstrate its successful application for segmentation of 2D and 3D electron and multicolor light microscopy datasets with isotropic and anisotropic voxels. We distribute DeepMIB as both an open-source multi-platform Matlab code and as compiled standalone application for Windows, MacOS and Linux. It comes in a single package that is simple to install and use as it does not require knowledge of programming. DeepMIB is suitable for everyone interested of bringing a power of deep learning into own image segmentation workflows. Author summary Deep learning approaches are highly sought after solutions for coping with large amounts of collected datasets and are expected to become an essential part of imaging workflows. However, in most cases, deep learning is still considered as a complex task that only image analysis experts can master. With DeepMIB we address this problem and provide the community with a user-friendly and open-source tool to train convolutional neural networks and apply them to segment 2D and 3D grayscale or multi-color datasets.Peer reviewe

Proton translocation coupled to electron transfer reactions in terminal oxidases

Terminal oxidases are the final proteins of the respiratory chain in eukaryotes and some bacteria. They catalyze most of the biological oxygen consumption on Earth done by aerobic organisms. During the catalytic reaction terminal oxidases reduce dioxygen to water and use the energy released in this process to maintain the electrochemical proton gradient by functioning as a redox-driven proton pump. This membrane gradient of protons is extremely important for cells as it is used for many cellular processes, such as transportation of substrates and ATP synthesis. Even though the structures of several terminal oxidases are known, they are not sufficient in themselves to explain the molecular mechanism of proton pumping. In this work we have applied a complex approach using a variety of different techniques to address the properties and the mechanism of proton translocation by the terminal oxidases. The combination of direct measurements of pH changes during catalytic turnover, time-resolved potentiometric electrometry and optical spectroscopy, made it possible to obtain valuable information about various aspects of oxidase functioning. We compared oxygen binding properties of terminal oxidases from the distinct heme-copper (CcO) and cytochrome bd families and found that cytochrome bd has a high affinity for oxygen, which is 3 orders of magnitude higher than that of CcO. Interestingly, the difference between CcO and cytochrome bd is not only in higher affinity of the latter to oxygen, but also in the way that each of these enzymes traps oxygen during catalysis. CcO traps oxygen kinetically - the molecule of bound dioxygen is rapidly reduced before it can dissociate. Alternatively, cytochrome bd employs an alternative mechanism of oxygen trapping - part of the redox energy is invested into tight oxygen binding, and the price paid for this is the lack of proton pumping. A single cycle of oxygen reduction to water is characterized by translocation of four protons across the membrane. Our results make it possible to assign the pumping steps to discrete transitions of the catalytic cycle and indicate that during in vivo turnover of the oxidase these four protons are transferred, one at a time, during the P→F, F→OH, Oh→Eh, and Eh→R transitions. At the same time, each individual proton translocation step in the catalytic cycle is not just a single reaction catalyzed by CcO, but rather a complicated sequence of interdependent electron and proton transfers. We assume that each single proton translocation cycle of CcO is assured by internal proton transfer from the conserved Glu-278 to an as yet unidentified pump site above the hemes. Delivery of a proton to the pump site serves as a driving reaction that forces the proton translocation cycle to continue.Terminaaliset oksidaasit ovat eukaryoottien ja joidenkin bakteerien hengitysketjujen viimeisiä proteiineja. Ne katalysoivat suurinta osaa aerobisten organismien biologisesta hapenkulutuksesta maapallolla. Katalyyttisen reaktion aikana terminaaliset oksidaasit pelkistävät molekulaarisen hapen vedeksi ja käyttävät prosessissa vapautuneen energian ylläpitääkseen elektrokemiallista protonigradienttia toimimalla protonipumppuina. Tämä protonien muodostama membraanigradientti on äärimmäisen tärkeä soluille, koska sitä käytetään hyväksi monissa solun toiminnoissa kuten substraattien kuljetuksessa ja ATP synteesissä. Vaikka useiden terminaalisten oksidaasien rakenteet tunnetaan, ne eivät itsessään riitä selittämään protonin pumppauksen molekulaarista mekanismia. Tässä työssä olemme käyttäneet monitahoista lähestymistapaa käyttäen erilaisia tekniikoita tutkiaksemme terminaalisten oksidaasien ominaisuuksia ja protonin pumppauksen mekanismia. Katalyyttisen reaktion aikana tapahtuvien pH:n muutosten mittaaminen sekä aikaerotteisen potentiometrisen elektrometrian ja optisen spektroskopian yhdistelmä mahdollisti arvokkaan tiedon keräämisen oksidaasien toiminnan eri osa-alueista. Me kykenimme vertailemaan terminaalisten oksidaasien hapensitomisominaisuuksia hemi-kuparioksidaasi ja bd-oksidaasi entsyymiperheiden välillä ja havaitsimme, että kyseiset proteiinit käyttävät erilaisia mekanismeja hapen sitomisessa. Hemi-kupari oksidaasit sitovat hapen kineettisesti - sitoutunut happimolekyyli pelkistetään nopeasti ennen kuin se ehtii irrota aktiivisesta keskuksesta, kun taas bd-oksidaasi käyttää hapetus-pelkistus energiaa hapen tiukkaan sitomiseen, ja on siten kykenemätön pumppaamaan protoneja. Yhden happimolekyylin pelkistäminen vedeksi mahdollistaa neljän protonin pumppaamisen kalvon yli. Tuloksemme mahdollistavat katalyyttisen kierron eri vaiheiden ja yksittäisten protoninpumppaus tapahtumien yhteen sovittamisen. Jokainen katalyyttisen kierron protonin pumppaus reaktio ei ole vain yksi entsyymin katalysoima reaktio, pikemminkin monimutkainen toisistaan riippuvaisten elektronin ja protonin siirtojen sarja. Oletamme, että jokainen protonin pumppaus reaktio varmistetaan proteiinin sisäisellä protoninsiirto reaktiolla vakioisesta glutamaatti tähteestä vielä tuntemattomaan pumppaus kohtaan . Protonin saapuminen pumppaus kohtaan toimii reaktion liikkeelle panevana voimana, joka pakottaa protonin pumppaus syklin jatkumaan

Time-resolved generation of membrane potential by ba3 cytochrome c oxidase from Thermus thermophilus coupled to single electron injection into the O and OH states

peer-reviewedTwo electrogenic phases with characteristic times of ~ 14 μs and ~ 290 μs are resolved in the kinetics of membrane potential generation coupled to single-electron reduction of the oxidized “relaxed” O state of ba3 oxidase from T. thermophilus (O → E transition). The rapid phase reflects electron redistribution between CuA and heme b. The slow phase includes electron redistribution from both CuA and heme b to heme a3, and electrogenic proton transfer coupled to reduction of heme a3. The distance of proton translocation corresponds to uptake of a proton from the inner water phase into the binuclear center where heme a3 is reduced, but there is no proton pumping and no reduction of CuB. Single-electron reduction of the oxidized “unrelaxed” state (OH → EH transition) is accompanied by electrogenic reduction of the heme b/heme a3 pair by CuA in a “fast” phase (~ 22 μs) and transfer of protons in “middle” and “slow” electrogenic phases (~ 0.185 ms and ~ 0.78 ms) coupled to electron redistribution from the heme b/heme a3 pair to the CuB site. The “middle” and “slow” electrogenic phases seem to be associated with transfer of protons to the proton-loading site (PLS) of the proton pump, but when all injected electrons reach CuB the electronic charge appears to be compensated by back-leakage of the protons from the PLS into the binuclear site. Thus proton pumping occurs only to the extent of ~ 0.1 H+/e−, probably due to the formed membrane potential in the experiment.ACCEPTEDpeer-reviewe

Automated 3D Axonal Morphometry of White Matter

Peer reviewe

DeepACSON automated segmentation of white matter in 3D electron microscopy

Tracing the entirety of ultrastructures in large three-dimensional electron microscopy (3D-EM) images of the brain tissue requires automated segmentation techniques. Current segmentation techniques use deep convolutional neural networks (DCNNs) and rely on high-contrast cellular membranes and high-resolution EM volumes. On the other hand, segmenting low-resolution, large EM volumes requires methods to account for severe membrane discontinuities inescapable. Therefore, we developed DeepACSON, which performs DCNN-based semantic segmentation and shape-decomposition-based instance segmentation. DeepACSON instance segmentation uses the tubularity of myelinated axons and decomposes under-segmented myelinated axons into their constituent axons. We applied DeepACSON to ten EM volumes of rats after sham-operation or traumatic brain injury, segmenting hundreds of thousands of long-span myelinated axons, thousands of cell nuclei, and millions of mitochondria with excellent evaluation scores. DeepACSON quantified the morphology and spatial aspects of white matter ultrastructures, capturing nanoscopic morphological alterations five months after the injury. With DeepACSON, Abdollahzadeh et al. combines existing deep learning-based methods for semantic segmentation and a novel shape decomposition technique for the instance segmentation. The pipeline is used to segment low-resolution 3D-EM datasets allowing quantification of white matter morphology in large fields-of-view.Peer reviewe

REEP3 and REEP4 determine the tubular morphology of the endoplasmic reticulum during mitosis

The endoplasmic reticulum (ER) is extensively remodeled during metazoan open mitosis. However, whether the ER becomes more tubular or more cisternal during mitosis is controversial, and dedicated factors governing the morphology of the mitotic ER have remained elusive. Here, we describe the ER membrane proteins REEP3 and REEP4 as major determinants of ER morphology in metaphase cells. REEP3/4 are specifically required for generating the high-curvature morphology of mitotic ER and promote ER tubulation through their reticulon homology domains (RHDs). This ER-shaping activity of REEP3/4 is distinct from their previously described function to clear ER from metaphase chromatin. We further show that related REEP proteins do not contribute to mitotic ER shaping and provide evidence that the REEP3/4 carboxyterminus mediates regulation of the proteins. These findings confirm that ER converts to higher curvature during mitosis, identify REEP3/4 as specific and crucial morphogenic factors mediating ER tubulation during mitosis, and define the first cell cycle-specific role for RHD proteins.Peer reviewe

gACSON software for automated segmentation and morphology analyses of myelinated axons in 3D electron microscopy

Background and Objective: Advances in electron microscopy (EM) now allow three-dimensional (3D) imaging of hundreds of micrometers of tissue with nanometer-scale resolution, providing new opportunities to study the ultrastructure of the brain. In this work, we introduce a freely available Matlab-based gACSON software for visualization, segmentation, assessment, and morphology analysis of myelinated axons in 3D-EM volumes of brain tissue samples. Methods: The software is equipped with a graphical user interface (GUI). It automatically segments the intra-axonal space of myelinated axons and their corresponding myelin sheaths and allows manual segmentation, proofreading, and interactive correction of the segmented components. gACSON analyzes the morphology of myelinated axons, such as axonal diameter, axonal eccentricity, myelin thickness, or gratio. Results: We illustrate the use of the software by segmenting and analyzing myelinated axons in six 3DEM volumes of rat somatosensory cortex after sham surgery or traumatic brain injury (TBI). Our results suggest that the equivalent diameter of myelinated axons in somatosensory cortex was decreased in TBI animals five months after the injury. Conclusion: Our results indicate that gACSON is a valuable tool for visualization, segmentation, assessment, and morphology analysis of myelinated axons in 3D-EM volumes. It is freely available at https://github.com/AndreaBehan/g-ACSON under the MIT license. (c) 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )Peer reviewe

Membrane potential stabilizes the O intermediate in liposomes containing bacteriorhodopsin

AbstractIn the bacteriorhodopsin-containing proteoliposomes, a laser flash is found to induce formation of a bathointermediate decaying in several seconds, the difference spectrum being similar to the purple–blue transition. Different pH buffers do not affect the intermediate, whereas an uncoupler, gramicidin A, and lipophilic ions accelerate decay of the intermediate or inhibit its formation. In the liposomes containing E204Q bacteriorhodopsin mutant, formation of the intermediate is suppressed. In the wild-type bacteriorhodopsin liposomes, the bathointermediate formation is pH-independent within the pH 5–7 range. The efficiency of the long-lived O intermediate formation increases at a low pH. In the wild-type as well as in the E204Q mutant purple membrane, the O intermediate decay is slowed down at slightly higher pH values than that of the purple–blue transition. It is suggested that the membrane potential affects the equilibrium between the bacteriorhodopsin ground state (Glu-204 is protonated and Asp-85 is deprotonated) and the O intermediate (Asp-85 is protonated and Glu-204 is deprotonated), stabilizing the latter by changing the relative affinity of Asp-85 and Glu-204 to H+. At a low pH, protonation of a proton-releasing group (possibly Glu-194) in the bacteriorhodopsin ground state seems to prevent deprotonation of the Glu-204 during the photocycle. Thus, all protonatable residues of the outward proton pathway should be protonated in the O intermediate. Under such conditions, membrane potential stabilization of the O intermediate in the liposomes can be attributed to the direct effect of the potential on the pK value of Asp-85

Specific inhibition of proton pumping by the T315V mutation in the K channel of cytochrome ba3 from Thermus thermophilus

Cytochrome ba3 from Thermus thermophilus belongs to the B family of heme‑copper oxidases and pumps protons across the membrane with an as yet unknown mechanism. The K channel of the A family heme‑copper oxidases provides delivery of a substrate proton from the internal water phase to the binuclear heme‑copper centre (BNC) during the reductive phase of the catalytic cycle, while the D channel is responsible for transferring both substrate and pumped protons. By contrast, in the B family oxidases there is no D-channel and the structural equivalent of the K channel seems to be responsible for the transfer of both categories of protons. Here we have studied the effect of the T315V substitution in the K channel on the kinetics of membrane potential generation coupled to the oxidative half-reaction of the catalytic cycle of cytochrome ba3. The results suggest that the mutated enzyme does not pump protons during the reaction of the fully reduced form with molecular oxygen in a single turnover. Specific inhibition of proton pumping in the T315V mutant appears to be a consequence of inability to provide rapid (τ ~ 100 μs) reprotonation of the internal transient proton donor(s) of the K channel. In contrast to the A family, the K channel of the B-type oxidases is necessary for the electrogenic transfer of both pumped and substrate protons during the oxidative half-reaction of the catalytic cycle.Peer reviewe