Organelle Positioning Research Articles

DLIC1, but not DLIC2, is upregulated in colon cancer and this contributes to proliferative overgrowth and migratory characteristics of cancer cells.

Cytoplasmic dynein-1 is a large minus-end-directed microtubule motor complex involved in membrane trafficking, organelle positioning, and microtubule organization. The roles of dynein light intermediate chains (DLICs; DLIC1 and DLIC2) within the complex are, however, still largely undefined. In this study, we investigated the possible roles of DLICs in epithelial homeostasis and colon cancer development. Mutant clonal analysis of Drosophila Dlic in the follicular epithelium of Drosophila ovary showed defects in nuclear positioning, epithelial integrity, and apical cell polarity. Consistently, knockdown of human DLIC1 and DLIC2 in colon carcinoma cells resulted in damaged epithelial organization, disturbed lumen formation, and impaired apical polarity establishment in three-dimensional cell culture. Depletion of DLIC1 and DLIC2 led to reduced proliferation, enhanced apoptosis rates, disrupted mitotic spindle assembly, and induction of G2/M arrest in cell cycle progression. Moreover, reduced levels of DLIC1 in contrast to DLIC2 impaired the migratory ability. On the other hand, immunohistochemical examination of human colorectal tissue samples and further colorectal cancer dataset analysis showed a significant upregulation for DLIC1 in tumors, whereas DLIC2 expression was unchanged. In addition, the overexpression of DLIC1 caused increased proliferation, decreased apoptosis and enhanced migration, whereas DLIC2 overexpression did not result in any significant changes. Together, these results indicate that DLIC1 and DLIC2 contribute to the establishment and maintenance of epithelial homeostasis. Furthermore, these findings present the first evidence that DLIC1 and DLIC2 have distinct roles in colon cancer development and that DLIC1 may contribute to proliferative overgrowth and migratory characteristics.

The type III intermediate filament vimentin regulates organelle distribution and modulates autophagy

The cytoskeletal protein vimentin plays a key role in positioning of organelles within the cytosol and has been linked to the regulation of numerous cellular processes including autophagy, however, how vimentin regulates autophagy remains relatively unexplored. Here we report that inhibition of vimentin using the steroidal lactone Withaferin A (WFA) causes vimentin to aggregate, and this is associated with the relocalisation of organelles including autophagosomes and lysosomes from the cytosol to a juxtanuclear location. Vimentin inhibition causes autophagosomes to accumulate, and we demonstrate this results from modulation of mechanistic target of rapamycin (mTORC1) activity, and disruption of autophagosome-lysosome fusion. We suggest that vimentin plays a physiological role in autophagosome and lysosome positioning, thus identifying vimentin as a key factor in the regulation of mTORC1 and autophagy.

Moss Kinesin-14 KCBP Accelerates Chromatid Motility in Anaphase.

KCBP is a microtubule (MT) minus-end-directed kinesin widely conserved in plants. It was shown in Arabidopsis that KCBP controls trichome cell shape by orchestrating MT and actin cytoskeletons using its tail and motor domains. In contrast, the KCBP knockout (KO) line in the moss Physcomitrella patens showed a defect in nuclear and organelle positioning in apical stem cells. Moss KCBP is postulated to transport the nucleus and chloroplast via direct binding to their membranes, since it binds to and transports liposomes composed of phospholipids in vitro. However, domains required for cargo transport in vivo have not been mapped. Here, we performed a structure-function analysis of moss KCBP. We found that the FERM domain in the tail region, which is known to bind to lipids as well as other proteins, is essential for both nuclear and chloroplast positioning, whereas the proximal MyTH4 domain plays a supporting role in chloroplast transport. After anaphase but prior to nuclear envelope re-formation, KCBP accumulates on the chromosomes, in particular at the centromeric region in a FERM-dependent manner. In the KCBP KO line, the rate of poleward chromosome movement in anaphase was reduced and lagging chromosomes occasionally appeared. These results suggest that KCBP binds to non-membranous naked chromosomes via an unidentified protein(s) for their transport. Finally, the liverwort orthologue of KCBP rescued the chromosome/chloroplast mis-positioning of the moss KCBP KO line, suggesting that the cargo transport function is conserved at least in bryophytes.Key words: kinesin, mitosis, chromosome segregation, kinetochore, dynein.

Cell organelle-based analysis of cell chirality

ABSTRACTThe maintenance of tight endothelial junctions requires the establishment of proper cell polarity, which includes not only the apicobasal and front-rear polarity but also the left-right (L-R) polarity. The cell possesses an intrinsic mechanism of orienting the L-R axis with respect to the other axes, following a left-hand or right-hand rule, termed cell chirality. We have previously reported that endothelial cells exhibit a clockwise or rightward bias on ring-shaped micropatterns. Now we further characterize the chirality of individual endothelial cells on micropatterns by analyzing the L-R positioning of the cell centroid relative to the nucleus-centrosome axis. Our results show that the centroids of endothelial cells preferably polarized towards the right side of the nucleus-centrosome axis. This bias is consistent with cell chirality characterized by other methods. These results suggest that the positioning of cell organelles is intrinsically L-R biased inside individual cells. This L-R bias provides an opportunity for determining cell chirality in situ, even in vivo, without the limitations of using isolated cells in in vitro engineered platforms.

Mitochondrial Organelle Movement Classification (Fission and Fusion) via Convolutional Neural Network Approach

Mitochondria are highly dynamic cellular organelles with the ability to change size, shape, and position over the course of a few seconds. Mitochondrial organelle movement refers to the problem of finding fission and fusion and generates energy for the cell. In this paper, we proposed a deep learning method [mitochondrial organelle movement classification (MOMC)] for mitochondrial movement classification using a convolutional neural network. We present a three-step feature description strategy, such as local descriptions, which is first extracted via the GoogLeNet, followed by the production of mid-level features by ResNet-50 and global descriptor features by Inception-V3 model and final classification of the position of mitochondrial organelle movement. Our method consists of a deep classification network, MOMC for gathering the organelle position, and a verification network for classification accuracy by removing false positives. Using machine learning methods, logistic regression (LR), support vector machine (SVM), and convolutional neural networks (CNNs), we found that the CNN better classified the shape of mitochondrial organelles (fission and fusion). Employing 24 types (position) of images, a convolutional neural network was trained to identify mitochondrial organelle movement with 96.32% accuracy. This enabled the discovery of position, further advancing the clinical utility of human mitochondrial organelles.

Achieving Automated Organelle Biopsy on Small Single Cells Using a Cell Surgery Robotic System.

Single cell surgery such as manipulation or removal of subcellular components or/and organelles from single cells is increasingly used for the study of diseases and their causes in precision medicine. This paper presents a robotic surgery system to achieve automated organelle biopsy of single cells with dimensions of less than 20μm in diameter. The complexity of spatial detection of the organelle position is reduced by patterning the cells using a microfluidic chip device. A sliding mode nonlinear controller is developed to enable extraction of organelles, such as the mitochondria and the nucleus, from single cells with high precision. An image processing algorithm is also developed to automatically detect the position of the desired organelle. The effectiveness of the proposed robotic surgery system is demonstrated experimentally with automated extraction of mitochondria and nucleus from human acute promyelocytic leukemia cells and human fibroblast cells. Extraction is followed by biological tests to indicate the functionality of biopsied mitochondria as well as the cell viability after removal of mitochondria. The results presented here have revealed that the proposed approach of automated organelle biopsy on single small cells is feasible.

Analysis of Organelle Positioning Using Patterned Microdevices.

The consequences of alterations in the distribution of intracellular organelles, observed in many diseases, are often not clear. Intracellular organelles alter their morphology and positioning to regulate cell homeostasis and function. We outline how organelle positioning can be studied employing a density-based analysis of 3D images applied to cells that show similar cellular geometries. Quantification is facilitated by the use of single cells seeded on micropatterned substrates that provide cues for controlled cell spreading. This minimal system mimics the reproducible distribution of organelles typically observed in tissues, simplifying image analysis and minimizing the number of cells required for the observation of robust phenotypes. Here we provide guidelines for how the majority of organelles can be efficiently analyzed in cells seeded on adhesive micropatterns. We exemplify how alterations in the positioning of different organelles as a result of the perturbation of the cytoskeleton or associated motor proteins can be efficiently quantified. © 2018 by John Wiley & Sons, Inc.

Determining the Intracellular Organization of Organelles.

Many studies have found alterations in the positioning and morphology of intracellular organelles under different experimental conditions. Although the precise quantification of these changes is challenging, it is strongly facilitated in single cells that are seeded on micropatterned substrates. Indeed, the controlled microenvironment of the cell leads to a reproducible distribution of organelles, simplifying image analysis and minimizing the number of cells required for robust phenotypes. Here, we outline how alterations in the intracellular organization of lysosomes and mitochondria, as a result of different growth conditions, can be efficiently quantified in cells seeded on adhesive micropatterns.

Vesicular trafficking plays a role in centriole disengagement and duplication.

Centrosomes are the major microtubule-nucleating and microtubule-organizing centers of cells and play crucial roles in microtubule anchoring, organelle positioning, and ciliogenesis. At the centrosome core lies a tightly associated or “engaged” mother–daughter centriole pair. During mitotic exit, removal of centrosomal proteins pericentrin and Cep215 promotes “disengagement” by the dissolution of intercentriolar linkers, ensuring a single centriole duplication event per cell cycle. Herein, we explore a new mechanism involving vesicular trafficking for the removal of centrosomal Cep215. Using small interfering RNA and CRISPR/Cas9 gene-edited cells, we show that the endocytic protein EHD1 regulates Cep215 transport from centrosomes to the spindle midbody, thus facilitating disengagement and duplication. We demonstrate that EHD1 and Cep215 interact and show that Cep215 displays increased localization to vesicles containing EHD1 during mitosis. Moreover, Cep215-containing vesicles are positive for internalized transferrin, demonstrating their endocytic origin. Thus, we describe a novel relationship between endocytic trafficking and the centrosome cycle, whereby vesicles of endocytic origin are used to remove key regulatory proteins from centrosomes to control centriole duplication.

A crystal structure of coil 1B of vimentin in the filamentous form provides a model of a high-order assembly of a vimentin filament.

Structural data are available in the PDB under the accession number 5WHF.

Genetically induced microtubule disruption in the mouse intestine impairs intracellular organization and transport.

In most differentiated cells, microtubules reorganize into noncentrosomal arrays that are cell-type specific. In the columnar absorptive enterocytes of the intestine, microtubules form polarized apical–basal arrays that have been proposed to play multiple roles. However, in vivo testing of these hypotheses has been hampered by a lack of genetic tools to specifically perturb microtubules. Here we analyze mice in which microtubules are disrupted by conditional inducible expression of the microtubule-severing protein spastin. Spastin overexpression resulted in multiple cellular defects, including aberrations in nuclear and organelle positioning and deficient nutrient transport. However, cell shape, adhesion, and polarity remained intact, and mutant mice continued to thrive. Notably, the phenotypes of microtubule disruption are similar to those induced by microtubule disorganization upon loss of CAMSAP3/Nezha. These data demonstrate that enterocyte microtubules have important roles in organelle organization but are not essential for growth under homeostatic conditions.

Tunable and Photoswitchable Chemically Induced Dimerization for Chemo‐optogenetic Control of Protein and Organelle Positioning

AbstractThe spatiotemporal dynamics of proteins and organelles play an important role in controlling diverse cellular processes. Optogenetic tools using photosensitive proteins and chemically induced dimerization (CID), which allow control of protein dimerization, have been used to elucidate the dynamics of biological systems and to dissect the complicated biological regulatory networks. However, the inherent limitations of current optogenetic and CID systems remain a significant challenge for the fine‐tuning of cellular activity at precise times and locations. Herein, we present a novel chemo‐optogenetic approach, photoswitchable chemically induced dimerization (psCID), for controlling cellular function by using blue light in a rapid and reversible manner. Moreover, psCID is tunable; that is, the dimerization and dedimerization degrees can be fine‐tuned by applying different doses of illumination. Using this approach, we control the localization of proteins and positioning of organelles in live cells with high spatial (μm) and temporal (ms) precision.

Tunable and Photoswitchable Chemically Induced Dimerization for Chemo-optogenetic Control of Protein and Organelle Positioning.

The spatiotemporal dynamics of proteins and organelles play an important role in controlling diverse cellular processes. Optogenetic tools using photosensitive proteins and chemically induced dimerization (CID), which allow control of protein dimerization, have been used to elucidate the dynamics of biological systems and to dissect the complicated biological regulatory networks. However, the inherent limitations of current optogenetic and CID systems remain a significant challenge for the fine‐tuning of cellular activity at precise times and locations. Herein, we present a novel chemo‐optogenetic approach, photoswitchable chemically induced dimerization (psCID), for controlling cellular function by using blue light in a rapid and reversible manner. Moreover, psCID is tunable; that is, the dimerization and dedimerization degrees can be fine‐tuned by applying different doses of illumination. Using this approach, we control the localization of proteins and positioning of organelles in live cells with high spatial (μm) and temporal (ms) precision.

P-130 - The conserved cysteine residue of type III intermediate filaments serves as a structural element and redox sensor

Glial fibrillary acidic protein (GFAP) along with vimentin, desmin and peripherin constitutes the type III class of intermediate filament proteins, which are expressed in a cell type-dependent manner. GFAP and vimentin play a role in organelle positioning, cell migration and mechanical resistance and possess a conserved cysteine residue (cys328 in vimentin and cys294 in GFAP). We previously showed that cys328 of vimentin is critical for stress sensing, since mutants lacking this residue are resistant to vimentin reorganization induced by oxidants and electrophilic lipids. We have observed that GFAP cys294 is also a target for lipoxidation, which induces a drastic filament rearrangement in cells. Therefore, the conserved cysteine residues of type III intermediate filaments emerge as important players in cellular responses to oxidative damage. Interestingly, cysteine to serine mutations compromise assembly and copolymerization of GFAP and vimentin, showing that these residues are important for the structure of the intermediate filament network. This raises the hypothesis that modifications of cysteine residues of intermediate filaments may contribute to cellular dysfunction under pathophysiological conditions.

Molecular model of the mitochondrial genome segregation machinery in Trypanosoma brucei

In almost all eukaryotes, mitochondria maintain their own genome. Despite the discovery more than 50 y ago, still very little is known about how the genome is correctly segregated during cell division. The protozoan parasite Trypanosoma brucei contains a single mitochondrion with a singular genome, the kinetoplast DNA (kDNA). Electron microscopy studies revealed the tripartite attachment complex (TAC) to physically connect the kDNA to the basal body of the flagellum and to ensure correct segregation of the mitochondrial genome via the basal bodies movement, during the cell cycle. Using superresolution microscopy, we precisely localize each of the currently known TAC components. We demonstrate that the TAC is assembled in a hierarchical order from the base of the flagellum toward the mitochondrial genome and that the assembly is not dependent on the kDNA itself. Based on the biochemical analysis, the TAC consists of several nonoverlapping subcomplexes, suggesting an overall size of the TAC exceeding 2.8 mDa. We furthermore demonstrate that the TAC is required for correct mitochondrial organelle positioning but not for organelle biogenesis or segregation.

Intracellular Fluid Mechanics: Coupling Cytoplasmic Flow with Active Cytoskeletal Gel

The cell is a mechanical machine, and continuum mechanics of the fluid cytoplasm and the viscoelastic deforming cytoskeleton play key roles in cell physiology. We review mathematical models of intracellular fluid mechanics, from cytoplasmic fluid flows, to the flow of a viscous active cytoskeletal gel, to models of two-phase poroviscous flows, to poroelastic models. We discuss application of these models to cell biological phenomena, such as organelle positioning, blebbing, and cell motility. We also discuss challenges of understanding fluid mechanics on the cellular scale.

Characterisation of maturation of photoreceptor cell subtypes during zebrafish retinal development.

ABSTRACTPhotoreceptor cells (PRCs) mature from simple epithelial cells, a process characterised by growth and compartmentalisation of the apical membrane into an inner and an outer segment. So far, a PRC subtype-specific description of morphological and cellular changes in the developing zebrafish retina is missing. Here, we performed an in-depth characterisation of four of the five PRC subtypes of the zebrafish retina between 51 and 120 h post fertilisation, including quantification of the size of different compartments, localisation of polarity proteins and positioning of organelles. One of the major findings was the anisotropic and subtype-specific growth of the different PRC compartments. In addition, a transient accumulation of endoplasmic reticulum in rod PRCs, changes in chromatin organisation in UV sensitive cones and differential expression of polarity proteins during the initial stages of PRC maturation were observed. The results obtained provide a developmental timeline that can be used as a platform for future studies on PRC maturation and function. This platform was applied to document that increased exposure to light leads to smaller apical domains of PRCs.

Rat liver intoxication with CCl4: biochemistry, histology, and mass spectrometry.

This work provides complex characterisation of cirrhotic rat liver tissue induced by carbon tetrachloride using biochemical and histopathological analyses, and also presents a novel approach, secondary ion mass spectrometry (SIMS). According to our knowledge, this is the first report that compares these three different approaches in study of liver damage. We observed increased levels of triacylglycerols and total cholesterol in the liver and decreased levels of those parameters in the plasma. Histopathological observations include fat accumulation in the cells and changes in internal configuration of cells such as shift of position of organelles from the centre to the edge. The damage to the rat tissue is additionally determined by SIMS analysis, which characterizes, among other substances, diacylglycerols, cholesterol and fatty acids, such as linoleic and oleic acids. Interestingly, unlike other observed particles, a marked difference in SIMS intensity for diacylglycerol C37H69O4 positive fragment at 575.5 m/u was observed. In fact, there was one order of magnitude difference between intoxicated liver samples and controls and this molecular signal seems to be a potential chemical indicator of the damage. The SIMS images are consistent with histopathological results and they additionally provide information about distribution of chemical compound which is a new potential tool for the liver disease characterisation on molecular level.

Functional Analysis of LINC Complexes in the Skin.

The genome in eukaryotic cells is encased by two intricate and interconnected concentric membranes, which together with the underlying nuclear lamina form the nuclear envelope (NE). Two fundamental macromolecular structures are embedded within the nuclear envelope: the nuclear pore (NPC) and the LINC complex. The former perforates the nucleus controlling biomolecule trafficking between the nucleoplasm and the cytoplasm, while the latter integrates the nucleus via the cytoskeleton to the extracellular matrix. LINC complex structural and functional integrity is of utmost importance for various fundamental cellular functions. Mechanical forces are relayed into the nuclear interior via the LINC complex, which controls lamina organization, chromosome dynamics, and genome organization and stability. Thus, LINC constituents play pivotal roles in cellular architecture including organelle positioning, cell movement, tissue assembly, organ homeostasis, and organismal aging. The LINC complex oligomeric core contains several multi-isomeric, multifunctional, and often tissue-specific proteins. Therefore, for a proper functional analysis, genetic mouse models are an invaluable resource. Herein, we focus on the LINC complex roles in the skin and describe methods that enable the successful isolation of primary embryonic fibroblast and newborn skin cells, which can be then investigated functionally in vitro.

Interaction between the flagellar pocket collar and the hook complex via a novel microtubule-binding protein in Trypanosoma brucei.

Trypanosoma brucei belongs to a group of unicellular, flagellated parasites that are responsible for human African trypanosomiasis. An essential aspect of parasite pathogenicity is cytoskeleton remodelling, which occurs during the life cycle of the parasite and is accompanied by major changes in morphology and organelle positioning. The flagellum originates from the basal bodies and exits the cell body through the flagellar pocket (FP) but remains attached to the cell body via the flagellum attachment zone (FAZ). The FP is an invagination of the pellicular membrane and is the sole site for endo- and exocytosis. The FAZ is a large complex of cytoskeletal proteins, plus an intracellular set of four specialised microtubules (MtQ) that elongate from the basal bodies to the anterior end of the cell. At the distal end of the FP, an essential, intracellular, cytoskeletal structure called the flagellar pocket collar (FPC) circumvents the flagellum. Overlapping the FPC is the hook complex (HC) (a sub-structure of the previously named bilobe) that is also essential and is thought to be involved in protein FP entry. BILBO1 is the only functionally characterised FPC protein and is necessary for FPC and FP biogenesis. Here, we used a combination of in vitro and in vivo approaches to identify and characterize a new BILBO1 partner protein—FPC4. We demonstrate that FPC4 localises to the FPC, the HC, and possibly to a proximal portion of the MtQ. We found that the C-terminal domain of FPC4 interacts with the BILBO1 N-terminal domain, and we identified the key amino acids required for this interaction. Interestingly, the FPC4 N-terminal domain was found to bind microtubules. Over-expression studies highlight the role of FPC4 in its association with the FPC, HC and FPC segregation. Our data suggest a tripartite association between the FPC, the HC and the MtQ.