New insights into the peroxisomal protein inventory: Acyl-CoA oxidases and -dehydrogenases are an ancient feature of peroxisomes.

Peroxisomes are ubiquitous organelles which participate in a variety of essential biochemical pathways. An intimate interrelationship between peroxisomes and mitochondria is emerging in mammals, where both organelles cooperate in fatty acid ß-oxidation and cellular lipid homeostasis. As mitochondrial fatty acid ß-oxidation is lacking in yeast and plants, suitable genetically accessible model systems to study this interrelationship are scarce. Here, we propose the filamentous fungus Ustilago maydis as a suitable model for those studies. We combined molecular cell biology, bioinformatics and phylogenetic analyses and provide the first comprehensive inventory of U. maydis peroxisomal proteins and pathways. Studies with a peroxisome-deficient Δpex3 mutant revealed the existence of parallel and complex, cooperative ß-oxidation pathways in peroxisomes and mitochondria, mimicking the situation in mammals. Furthermore, we provide evidence that acyl-CoA dehydrogenases (ACADs) are bona fide peroxisomal proteins in fungi and mammals and together with acyl-CoA oxidases (ACOX) belong to the basic enzymatic repertoire of peroxisomes. A genome comparison with baker's yeast and human gained new insights into the basic peroxisomal protein inventory shared by humans and fungi and revealed novel peroxisomal proteins and functions in U. maydis. The importance of our findings for the evolution and function of the complex interrelationship between peroxisomes and mitochondria in fatty acid ß-oxidation is discussed.

Pex11pbeta-mediated growth and division of mammalian peroxisomes follows a maturation pathway.

Peroxisomes are ubiquitous subcellular organelles, which multiply by growth and division but can also form de novo via the endoplasmic reticulum. Growth and division of peroxisomes in mammalian cells involves elongation, membrane constriction and final fission. Dynamin-like protein (DLP1/Drp1) and its membrane adaptor Fis1 function in the later stages of peroxisome division, whereas the membrane peroxin Pex11pbeta appears to act early in the process. We have discovered that a Pex11pbeta-YFP(m) fusion protein can be used as a specific tool to further dissect peroxisomal growth and division. Pex11pbeta-YFP(m) inhibited peroxisomal segmentation and division, but resulted in the formation of pre-peroxisomal membrane structures composed of globular domains and tubular extensions. Peroxisomal matrix and membrane proteins were targeted to distinct regions of the peroxisomal structures. Pex11pbeta-mediated membrane formation was initiated at pre-existing peroxisomes, indicating that growth and division follows a multistep maturation pathway and that formation of mammalian peroxisomes is more complex than simple division of a pre-existing organelle. The implications of these findings on the mechanisms of peroxisome formation and membrane deformation are discussed.

A paradigm shift: Bioengineering meets mechanobiology towards overcoming remyelination failure.

Therapeutic strategies aimed at overcoming the loss of myelin sheath in central nervous system demyelinating diseases are often unsuccessful due to nescience underlying the mechanisms of remyelination failure. The environment surrounding a demyelination lesion is seen as a hostile terrain, characterized by factors that can inhibit myelin production by oligodendrocytes (OLs). The formation of a glial scar containing reactive astrocytes producing high amounts of altered matrix proteins can compromise OL remyelination. Allied to glial scar, mechanical properties of the tissue are altered. The paradigms in the remyelination failure are changing. We point mechanobiology as a missing key towards unravelling the nature of (de)myelination. Mechanical cues as stiffness, axonal tension or physical constraints are emerging as dictators of tissue homeostasis and pathology. Here we delve into an in-depth characterization of the preeminent models to study mechanobiology events of (de)myelination and remyelination. Alternatives to in vivo systems are provided, either through the exploration of simpler animal models, creation of in vitro models using tissue engineered approaches or through in silico tools. We discuss how bioengineering is being explored to generate relevant models to dissect new mechanobiology mechanisms and identify novel therapeutic targets, being expected to profoundly impact the treatment of demyelinating diseases.

Labeling of Peroxisomes for Live Cell Imaging in the Filamentous Fungus Ustilago maydis.

The basidiomycete fungus Ustilago maydis has emerged as a powerful model organism to study fundamental biological processes. U. maydis shares many important features with human cells but provides the technical advantages of yeast. Recently, U. maydis has also been used to investigate fundamental processes in peroxisome biology. Here, we present an efficient yeast recombination-based cloning method to construct and express fluorescent fusion proteins (or conditional mutant protein alleles) which target peroxisomes in the fungus U. maydis. In vivo analysis is pivotal for understanding the underlying mechanisms of organelle motility. We focus on the in vivo labeling of peroxisomes in U. maydis and present approaches to analyze peroxisomal motility.

Biological Implications of Differential Expression of Mitochondrial-Shaping Proteins in Parkinson's Disease.

It has long been accepted that mitochondrial function and morphology is affected in Parkinson's disease, and that mitochondrial function can be directly related to its morphology. So far, mitochondrial morphological alterations studies, in the context of this neurodegenerative disease, have been performed through microscopic methodologies. The goal of the present work is to address if the modifications in the mitochondrial-shaping proteins occurring in this disorder have implications in other cellular pathways, which might constitute important pathways for the disease progression. To do so, we conducted a novel approach through a thorough exploration of the available proteomics-based studies in the context of Parkinson's disease. The analysis provided insight into the altered biological pathways affected by changes in the expression of mitochondrial-shaping proteins via different bioinformatic tools. Unexpectedly, we observed that the mitochondrial-shaping proteins altered in the context of Parkinson's disease are, in the vast majority, related to the organization of the mitochondrial cristae. Conversely, in the studies that have resorted to microscopy-based techniques, the most widely reported alteration in the context of this disorder is mitochondria fragmentation. Cristae membrane organization is pivotal for mitochondrial ATP production, and changes in their morphology have a direct impact on the organization and function of the oxidative phosphorylation (OXPHOS) complexes. To understand which biological processes are affected by the alteration of these proteins we analyzed the binding partners of the mitochondrial-shaping proteins that were found altered in Parkinson's disease. We showed that the binding partners fall into seven different cellular components, which include mitochondria, proteasome, and endoplasmic reticulum (ER), amongst others. It is noteworthy that, by evaluating the biological process in which these modified proteins are involved, we showed that they are related to the production and metabolism of ATP, immune response, cytoskeleton alteration, and oxidative stress, amongst others. In summary, with our bioinformatics approach using the data on the modified proteins in Parkinson's disease patients, we were able to relate the alteration of mitochondrial-shaping proteins to modifications of crucial cellular pathways affected in this disease.

Peroxisomes, lipid droplets, and endoplasmic reticulum "hitchhike" on motile early endosomes.

Intracellular transport is mediated by molecular motors that bind cargo to be transported along the cytoskeleton. Here, we report, for the first time, that peroxisomes (POs), lipid droplets (LDs), and the endoplasmic reticulum (ER) rely on early endosomes (EEs) for intracellular movement in a fungal model system. We show that POs undergo kinesin-3- and dynein-dependent transport along microtubules. Surprisingly, kinesin-3 does not colocalize with POs. Instead, the motor moves EEs that drag the POs through the cell. PO motility is abolished when EE motility is blocked in various mutants. Most LD and ER motility also depends on EE motility, whereas mitochondria move independently of EEs. Covisualization studies show that EE-mediated ER motility is not required for PO or LD movement, suggesting that the organelles interact with EEs independently. In the absence of EE motility, POs and LDs cluster at the growing tip, whereas ER is partially retracted to subapical regions. Collectively, our results show that moving EEs interact transiently with other organelles, thereby mediating their directed transport and distribution in the cell.