MUCKE aims to mine a large volume of images, to structure them conceptually and to use this conceptual structuring in order to improve large-scale image retrieval. The last decade witnessed important progress concerning low-level image representations. However, there are a number problems which need to be solved in order to unleash the full potential of image mining in applications. The central problem with low-level representations is the mismatch between them and the human interpretation of image content. This problem can be instantiated, for instance, by the incapability of existing descriptors to capture spatial relationships between the concepts represented or by their incapability to convey an explanation of why two images are similar in a content-based image retrieval framework. We start by assessing existing local descriptors for image classification and by proposing to use co-occurrence matrices to better capture spatial relationships in images. The main focus in MUCKE is on cleaning large scale Web image corpora and on proposing image representations which are closer to the human interpretation of images. Consequently, we introduce methods which tackle these two problems and compare results to state of the art methods. Note: some aspects of this deliverable are withheld at this time as they are pending review. Please contact the authors for a preview.

http://ieeexplore.ieee.org/iel2/4524/12820/00592460.pdf

Image Processing

We present ilastik, an easy-to-use interactive tool that brings machine-learning-based (bio)image analysis to end users without substantial computational expertise. It contains pre-defined workflows for image segmentation, object classification, counting and tracking. Users adapt the workflows to the problem at hand by interactively providing sparse training annotations for a nonlinear classifier. ilastik can process data in up to five dimensions (3D, time and number of channels). Its computational back end runs operations on-demand wherever possible, allowing for interactive prediction on data larger than RAM. Once the classifiers are trained, ilastik workflows can be applied to new data from the command line without further user interaction. We describe all ilastik workflows in detail, including three case studies and a discussion on the expected performance.

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ilastik: interactive machine learning for (bio)image analysis.

Lipid droplets are storage organelles at the centre of lipid and energy homeostasis. They have a unique architecture consisting of a hydrophobic core of neutral lipids, which is enclosed by a phospholipid monolayer that is decorated by a specific set of proteins. Originating from the endoplasmic reticulum, lipid droplets can associate with most other cellular organelles through membrane contact sites. It is becoming apparent that these contacts between lipid droplets and other organelles are highly dynamic and coupled to the cycles of lipid droplet expansion and shrinkage. Importantly, lipid droplet biogenesis and degradation, as well as their interactions with other organelles, are tightly coupled to cellular metabolism and are critical to buffer the levels of toxic lipid species. Thus, lipid droplets facilitate the coordination and communication between different organelles and act as vital hubs of cellular metabolism.

Dynamics and functions of lipid droplets

The endoplasmic reticulum (ER) is a large, dynamic structure that serves many roles in the cell including calcium storage, protein synthesis and lipid metabolism. The diverse functions of the ER are performed by distinct domains; consisting of tubules, sheets and the nuclear envelope. Several proteins that contribute to the overall architecture and dynamics of the ER have been identified, but many questions remain as to how the ER changes shape in response to cellular cues, cell type, cell cycle state and during development of the organism. Here we discuss what is known about the dynamics of the ER, what questions remain, and how coordinated responses add to the layers of regulation in this dynamic organelle.

/pdf/the-endoplasmic-reticulum-structure-function-and-response-to-46s4vmf7yp.pdf

The endoplasmic reticulum: structure, function and response to cellular signaling

UPR, autophagy, and mitochondria crosstalk underlies the ER stress response

Even with the assistance of many cellular factors, a significant fraction of newly synthesized proteins ends up misfolded. Cells evolved protein quality control systems to ensure that these potentially toxic species are detected and eliminated. The best characterized of these pathways, the ER-associated protein degradation (ERAD), monitors the folding of membrane and secretory proteins whose biogenesis takes place in the endoplasmic reticulum (ER). There is also increasing evidence that ERAD controls other ER-related functions through regulated degradation of certain folded ER proteins, further highlighting the role of ERAD in cellular homeostasis.

/pdf/er-associated-degradation-protein-quality-control-and-beyond-4a11r9la5i.pdf

ER-associated degradation: Protein quality control and beyond

We have characterized the requirements to inhibit the function of the plant vacuolar sorting receptor BP80 in vivo and gained insight into the crucial role of receptor recycling between the prevacuolar compartment and the Golgi apparatus The drug wortmannin interferes with the BP80-mediated route to the vacuole and induces hypersecretion of a soluble BP80-ligand Wortmannin does not prevent receptor-ligand binding itself but causes BP80 levels to be limiting Consequently, overexpression of BP80 partially restores vacuolar cargo transport To simulate receptor traffic, we tested a truncated BP80 derivative in which the entire lumenal domain of BP80 has been replaced by the green fluorescent protein (GFP) The resulting chimeric protein (GFP-BP80) accumulates in the prevacuolar compartment as expected, but a soluble GFP fragment can also be detected in purified vacuoles Interestingly, GFP-BP80 coexpression interferes with the correct sorting of a BP80-ligand and causes hypersecretion that is reversible by expressing a 10-fold excess of full-length BP80 This suggests that GFP-BP80 competes with endogenous BP80 mainly at the retrograde transport route that rescues receptors from the prevacuolar compartment Treatment with wortmannin causes further leakage of GFP-BP80 from the prevacuolar compartment to the vacuoles, whereas BP80-ligands are secreted We propose that recycling of the vacuolar sorting receptor from the prevacuolar compartment to the Golgi apparatus is an essential process that is saturable and wortmannin sensitive

Receptor salvage from the prevacuolar compartment is essential for efficient vacuolar protein targeting.

Sterol homeostasis is essential for the function of cellular membranes and requires feedback inhibition of HMGR, a rate-limiting enzyme of the mevalonate pathway As HMGR acts at the beginning of the pathway, its regulation affects the synthesis of sterols and of other essential mevalonate-derived metabolites, such as ubiquinone or dolichol Here, we describe a novel, evolutionarily conserved feedback system operating at a sterol-specific step of the mevalonate pathway This involves the sterol-dependent degradation of squalene monooxygenase mediated by the yeast Doa10 or mammalian Teb4, a ubiquitin ligase implicated in a branch of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway Since the other branch of ERAD is required for HMGR regulation, our results reveal a fundamental role for ERAD in sterol homeostasis, with the two branches of this pathway acting together to control sterol biosynthesis at different levels and thereby allowing independent regulation of multiple products of the mevalonate pathway

/pdf/sterol-homeostasis-requires-regulated-degradation-of-413t14evmb.pdf

Sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase Doa10/Teb4

Misfolded proteins in the endoplasmic reticulum (ER) are eliminated by a quality control system called ER-associated protein degradation (ERAD). However, it is unknown how misfolded proteins in the inner nuclear membrane (INM), a specialized ER subdomain, are degraded. We used a quantitative proteomics approach to reveal an ERAD branch required for INM protein quality control in yeast. This branch involved the integral membrane proteins Asi1, Asi2, and Asi3, which assembled into an Asi complex. Besides INM misfolded proteins, the Asi complex promoted the degradation of functional regulators of sterol biosynthesis. Asi-mediated ERAD was required for ER homeostasis, which suggests that spatial segregation of protein quality control systems contributes to ER function.

https://science.sciencemag.org/content/sci/346/6210/751.full.pdf

Quality control of inner nuclear membrane proteins by the Asi complex

V Malhotra is an Institucio Catalana de Recerca i Estudis Avancats professor at the Centre for Genomic Regulation, the work in his laboratory is funded by grants from the Ministerio de Economia, Industria y Competitividad Plan Nacional (ref. BFU2013-44188-P) and Consolider (CSD2009-00016). We acknowledge support of the Spanish Ministry of Economy and Competitiveness, through the Programmes ‘Centro de Excelencia Severo Ochoa 2013–2017’ (SEV-2012–0208) and Maria de Maeztu Units of Excellence in R and D (MDM-2015–0502). We acknowledge the support of the CERCA Programme/Generalitat de Catalunya. F Campelo and M Garcia-Parajo acknowledge support by the Spanish Ministry of Economy and Competitiveness (‘Severo Ochoa’ Programme for Centres of Excellence in R and D (SEV-2015–240522) and FIS2014-56107-R), BFU2015-73288-JIN, AEI/FEDER; UE, Fundacion Privada Cellex, HFSP (GA RGP0027/2012), EC FP7-NANO-VISTA (GA 288263) and LaserLab 4 Europe (GA 654148). I. Raote and F. Campelo acknowledge support by the BIST Ignite Grant (eTANGO).

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Ombretta Foresti

Papers

ER-associated degradation: Protein quality control and beyond

Receptor salvage from the prevacuolar compartment is essential for efficient vacuolar protein targeting.

Sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase Doa10/Teb4

Quality control of inner nuclear membrane proteins by the Asi complex

TANGO1 builds a machine for collagen export by recruiting and spatially organizing COPII, tethers and membranes