Aleksandr Mironov

Dynein is required for receptor sorting and the morphogenesis of early endosomes

The early endosome is organised into domains to ensure the separation of cargo. Activated mitogenic receptors, such as epidermal growth factor (EGF) receptor, are concentrated into vacuoles enriched for the small GTPase Rab5, which progressively exclude nutrient receptors, such as transferrin receptor, into neighbouring tubules. These vacuoles become enlarged, increase their content of intralumenal vesicles as EGF receptor is sorted from the limiting membrane, and eventually mature to late endosomes. Maturation is governed by the loss of Rab5 and is accompanied by the movement of endosomes along microtubules towards the cell centre. Here, we show that EGF relocates to the cell centre in a dynein-dependent fashion, concomitant with the sorting away of transferrin receptor, although it remains in Rab5-positive early endosomes. When dynein function is acutely disrupted, efficient recycling of transferrin from EGF-containing endosomes is retarded, loss of Rab5 is slowed and endosome enlargement is reduced.

Using transmission electron microscopy and 3View to determine collagen fibril size and three-dimensional organization

Collagen fibrils are the major tensile element in vertebrate tissues, in which they occur as ordered bundles in the extracellular matrix. Abnormal fibril assembly and organization results in scarring, fibrosis, poor wound healing and connective tissue diseases. Transmission electron microscopy (TEM) is used to assess the formation of the fibrils, predominantly by measuring fibril diameter. Here we describe a protocol for measuring fibril diameter as well as fibril volume fraction, mean fibril length, fibril cross-sectional shape and fibril 3D organization, all of which are major determinants of tissue function. Serial-section TEM (ssTEM) has been used to visualize fibril 3D organization in vivo. However, serial block face–scanning electron microscopy (SBF-SEM) has emerged as a time-efficient alternative to ssTEM. The protocol described below is suitable for preparing tissues for TEM and SBF-SEM (by 3View). We describe how to use 3View for studying collagen fibril organization in vivo and show how to find and track individual fibrils. The overall time scale is ∼8 d from isolating the tissue to having a 3D image stack.

The Bro1-related protein HD-PTP/PTPN23 is required for endosomal cargo sorting and multivesicular body morphogenesis.

The Saccharomyces cerevisiae protein Bro1p is required for sorting endocytic cargo to the lumen of multivesicular bodies (MVBs). The mammalian ortholog of Bro1p is not known; although Alix, a structurally related protein, supports the topologically similar process of virus budding, functional studies have so far failed to identify a role for Alix in MVB formation. To establish whether Alix or similar protein(s) participate in endosomal sorting, we attached a retroviral peptide that binds Alix to a reporter receptor. This chimera was sorted efficiently away from the early endosome to the lumen of late endocytic compartments. Surprisingly, sorting was not prevented by depleting Alix but instead required the Alix-related protein His domain phosphotyrosine phosphatase (HD-PTP)/His-Domain/Type N23 protein tyrosine phosphatase (PTPN23). Depletion of HD-PTP also reduced transfer of fluid-phase markers and EGF receptor to lysosomes, caused the accumulation of ubiquitinated proteins on endosomal compartments and disrupted the morphogenesis of MVBs. Rescue experiments using an RNAi-resistant version of HD-PTP and HD-PTP mutants demonstrated an essential role for the HD-PTP Bro1 domain, with ESCRT-III binding correlating with full biological activity.

Differential Regulation of Elastic Fiber Formation by Fibulin-4 and -5

Fibulin-4 and -5 are extracellular glycoproteins with essential non-compensatory roles in elastic fiber assembly. We have determined how they interact with tropoelastin, lysyl oxidase, and fibrillin-1, thereby revealing how they differentially regulate assembly. Strong binding between fibulin-4 and lysyl oxidase enhanced the interaction of fibulin-4 with tropoelastin, forming ternary complexes that may direct elastin cross-linking. In contrast, fibulin-5 did not bind lysyl oxidase strongly but bound tropoelastin in terminal and central regions and could concurrently bind fibulin-4. Both fibulins differentially bound N-terminal fibrillin-1, which strongly inhibited their binding to lysyl oxidase and tropoelastin. Knockdown experiments revealed that fibulin-5 controlled elastin deposition on microfibrils, although fibulin-4 can also bind fibrillin-1. These experiments provide a molecular account of the distinct roles of fibulin-4 and -5 in elastic fiber assembly and how they act in concert to chaperone cross-linked elastin onto microfibrils.

Glomerular Cell Cross-Talk Influences Composition and Assembly of Extracellular Matrix

The glomerular basement membrane (GBM) is a specialized extracellular matrix (ECM) compartment within the glomerulus that contains tissue-restricted isoforms of collagen IV and laminin. It is integral to the capillary wall and therefore, functionally linked to glomerular filtration. Although the composition of the GBM has been investigated with global and candidate-based approaches, the relative contributions of glomerular cell types to the production of ECM are not well understood. To characterize specific cellular contributions to the GBM, we used mass spectrometry-based proteomics to analyze ECM isolated from podocytes and glomerular endothelial cells in vitro. These analyses identified cell type-specific differences in ECM composition, indicating distinct contributions to glomerular ECM assembly. Coculture of podocytes and endothelial cells resulted in an altered composition and organization of ECM compared with monoculture ECMs, and electron microscopy revealed basement membrane-like ECM deposition between cocultured cells, suggesting the involvement of cell-cell cross-talk in the production of glomerular ECM. Notably, compared with monoculture ECM proteomes, the coculture ECM proteome better resembled a tissue-derived glomerular ECM dataset, indicating its relevance to GBM in vivo. Protein network analyses revealed a common core of 35 highly connected structural ECM proteins that may be important for glomerular ECM assembly. Overall, these findings show the complexity of the glomerular ECM and suggest that both ECM composition and organization are context-dependent.

Collagen XXVII is developmentally regulated and forms thin fibrillar structures distinct from those of classical vertebrate fibrillar collagens.

We have generated an antiserum to the variable domain of mouse collagen XXVII, a recently discovered novel member of the fibrillar collagen family. Collagen XXVII protein was first detectable in the mouse at embryonic day 12.5 (E12.5). By E14.5, the protein localized to cartilage, developing dermis, cornea, the inner limiting membrane of the retina, and major arteries of the heart. However, at E18.5, collagen XXVII protein was no longer apparent in most tissues and appeared restricted mainly to cartilage where expression continued into adulthood. Type XXVII collagen immunolocalized to 10-nm-thick nonstriated fibrils that were distinct from fibrils formed by the classical fibrillar collagens. The transient nature of its expression and unusual fibrillar structure suggest that collagen XXVII plays a developmental role distinct from those of the classical fibrillar collagens.

Nonmuscle myosin II powered transport of newly formed collagen fibrils at the plasma membrane

Significance Collagen is the most abundant protein in vertebrates and is the building block of strong tissues such as tendons, skin, and bones. The fibrils can be millimeters long and occur in the extracellular matrix as a scaffold for tissue growth. Important questions remain unanswered about how cells assemble and transport the fibrils. We show here that collagen fibril assembly can occur at the plasma membrane in structures called fibripositors. We show that fibripositors are a nonmuscle myosin II (NMII)-dependent mechanical interface between the actinomyosin machinery and the extracellular matrix; thus, we propose a new function for NMII. A unique mechanism of fibril transport is presented as a basis for studies of tissue morphogenesis and conditions including wound healing and fibrosis. Collagen fibrils can exceed thousands of microns in length and are therefore the longest, largest, and most size-pleomorphic protein polymers in vertebrates; thus, knowing how cells transport collagen fibrils is essential for a more complete understanding of protein transport and its role in tissue morphogenesis. Here, we identified newly formed collagen fibrils being transported at the surface of embryonic tendon cells in vivo by using serial block face-scanning electron microscopy of the cell-matrix interface. Newly formed fibrils ranged in length from ∼1 to ∼30 µm. The shortest (1–10 µm) occurred in intracellular fibricarriers; the longest (∼30 µm) occurred in plasma membrane fibripositors. Fibrils and fibripositors were reduced in numbers when collagen secretion was blocked. ImmunoEM showed the absence of lysosomal-associated membrane protein 2 on fibricarriers and fibripositors and there was no effect of leupeptin on fibricarrier or fibripositor number and size, suggesting that fibricarriers and fibripositors are not part of a fibril degradation pathway. Blebbistatin decreased fibricarrier number and increased fibripositor length; thus, nonmuscle myosin II (NMII) powers the transport of these compartments. Inhibition of dynamin-dependent endocytosis with dynasore blocked fibricarrier formation and caused accumulation of fibrils in fibripositors. Data from fluid-phase HRP electron tomography showed that fibricarriers could originate at the plasma membrane. We propose that NMII-powered transport of newly formed collagen fibrils at the plasma membrane is fundamental to the development of collagen fibril-rich tissues. A NMII-dependent cell-force model is presented as the basis for the creation and dynamics of fibripositor structures.

A mutation in the mouse Amelx tri-tyrosyl domain results in impaired secretion of amelogenin and phenocopies human X-linked amelogenesis imperfecta

Amelogenesis imperfecta (AI) describes a broad group of clinically and genetically heterogeneous inherited defects of dental enamel bio-mineralization. Despite identification of a number of genetic mutations underlying AI, the precise causal mechanisms have yet to be determined. Using a multi-disciplinary approach, we describe here a mis-sense mutation in the mouse Amelx gene resulting in a Y → H substitution in the tri-tyrosyl domain of the enamel extracellular matrix protein amelogenin. The enamel in affected animals phenocopies human X-linked AI where similar mutations have been reported. Animals affected by the mutation have severe defects of enamel bio-mineralization associated with absence of full-length amelogenin protein in the developing enamel matrix, loss of ameloblast phenotype, increased ameloblast apoptosis and formation of multi-cellular masses. We present evidence to demonstrate that affected ameloblasts express but fail to secrete full-length amelogenin leading to engorgement of the endoplasmic reticulum/Golgi apparatus. Immunohistochemical analysis revealed accumulations of both amelogenin and ameloblastin in affected cells. Co-transfection of Ambn and mutant Amelx in a eukaryotic cell line also revealed intracellular abnormalities and increased cytotoxicity compared with cells singly transfected with wild-type Amelx, mutant Amelx or Ambn or co-transfected with both wild-type Amelx and Ambn. We hypothesize that intracellular protein–protein interactions mediated via the amelogenin tri-tyrosyl motif are a key mechanistic factor underpinning the molecular pathogenesis in this example of AI. This study therefore successfully links phenotype with underlying genetic lesion in a relevant murine model for human AI.

Heimler Syndrome Is Caused by Hypomorphic Mutations in the Peroxisome-Biogenesis Genes PEX1 and PEX6

Heimler syndrome (HS) is a rare recessive disorder characterized by sensorineural hearing loss (SNHL), amelogenesis imperfecta, nail abnormalities, and occasional or late-onset retinal pigmentation. We ascertained eight families affected by HS and, by using a whole-exome sequencing approach, identified biallelic mutations in PEX1 or PEX6 in six of them. Loss-of-function mutations in both genes are known causes of a spectrum of autosomal-recessive peroxisome-biogenesis disorders (PBDs), including Zellweger syndrome. PBDs are characterized by leukodystrophy, hypotonia, SNHL, retinopathy, and skeletal, craniofacial, and liver abnormalities. We demonstrate that each HS-affected family has at least one hypomorphic allele that results in extremely mild peroxisomal dysfunction. Although individuals with HS share some subtle clinical features found in PBDs, the diagnosis was not suggested by routine blood and skin fibroblast analyses used to detect PBDs. In conclusion, our findings define HS as a mild PBD, expanding the pleiotropy of mutations in PEX1 and PEX6.

A 125 kDa RNase E/G-like protein is present in plastids and is essential for chloroplast development and autotrophic growth in Arabidopsis

Endoribonuclease E (RNase E) is a regulator of global gene expression in Escherichia coli and is the best studied member of the RNase E/G ribonuclease family. Homologues are present in other bacteria but the roles of plant RNase E/G-like proteins are not known. Arabidopsis thaliana contains a single nuclear gene (At2g04270) encoding a product with the conserved catalytic domain of RNase E/G-like proteins. At2g04270 and the adjacent At2g04280 gene form converging transcription units with a ∼40 base overlap at their 3’ ends. Several translation products were predicted from the analyses of At2g04270 cDNAs. An antibody raised against a recombinant A. thaliana RNase E/G-like protein recognized a 125 kDa protein band in purified chloroplast preparations fractionated by SDS-PAGE. The 125 kDa RNase E/G-like protein was detected in cotyledons, rosette and cauline leaves. T-DNA insertions in exon 6 or intron 11 of At2g04270 result in loss of the 125 kDa band or truncation to a 110 kDa band. Loss of At2g04270 function resulted in the arrest of chloroplast development, loss of autotrophic growth, and reduced plastid ribosomal, psbA and rbcL RNA levels. Homozygous mutant plants were pale-green, contained smaller plastids with fewer thylakoids and shorter granal stacks than wild-type chloroplasts, and required sucrose at all growth stages following germination right up to flowering and setting seeds. Recombinant A. thaliana RNase E/G-like proteins rescued an E. coli RNase E mutant and cleaved an rbcL RNA substrate. Expression of At2g04270 was highly correlated with genes encoding plastid polyribonucleotide phosphorylase, S1 RNA-binding, and CRS1/YhbY domain proteins.