| 1. |
- Bekkhus, Tove, et al.
(författare)
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Automated detection of vascular remodeling in human tumor draining lymph nodes by the deep learning tool HEV-finder
- 2022
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Ingår i: Journal of Pathology. - : John Wiley & Sons. - 0022-3417 .- 1096-9896. ; 258:1, s. 4-11
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Tidskriftsartikel (refereegranskat)abstract
- Vascular remodeling is common in human cancer and has potential as future biomarkers for prediction of disease progression and tumor immunity status. It can also affect metastatic sites, including the tumor-draining lymph nodes (TDLNs). Dilation of the high endothelial venules (HEVs) within TDLNs has been observed in several types of cancer. We recently demonstrated that it is a premetastatic effect that can be linked to tumor invasiveness in breast cancer. Manual visual assessment of changes in vascular morphology is a tedious and difficult task, limiting high-throughput analysis. Here we present a fully automated approach for detection and classification of HEV dilation. By using 12,524 manually classified HEVs, we trained a deep-learning model and created a graphical user interface for visualization of the results. The tool, named the HEV-finder, selectively analyses HEV dilation in specific regions of the lymph nodes. We evaluated the HEV-finder's ability to detect and classify HEV dilation in different types of breast cancer compared to manual annotations. Our results constitute a successful example of large-scale, fully automated, and user-independent, image-based quantitative assessment of vascular remodeling in human pathology and lay the ground for future exploration of HEV dilation in TDLNs as a biomarker.
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| 2. |
- Fabry, Ben, et al.
(författare)
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Focal adhesion kinase stabilizes the cytoskeleton.
- 2011
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 101:9, s. 2131-8
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Tidskriftsartikel (refereegranskat)abstract
- Focal adhesion kinase (FAK) is a central focal adhesion protein that promotes focal adhesion turnover, but the role of FAK for cell mechanical stability is unknown. We measured the mechanical properties of wild-type (FAKwt), FAK-deficient (FAK-/-), FAK-silenced (siFAK), and siControl mouse embryonic fibroblasts by magnetic tweezer, atomic force microscopy, traction microscopy, and nanoscale particle tracking microrheology. FAK-deficient cells showed lower cell stiffness, reduced adhesion strength, and increased cytoskeletal dynamics compared to wild-type cells. These observations imply a reduced stability of the cytoskeleton in FAK-deficient cells. We attribute the reduced cytoskeletal stability to rho-kinase activation in FAK-deficient cells that suppresses the formation of ordered stress fiber bundles, enhances cortical actin distribution, and reduces cell spreading. In agreement with this interpretation is that cell stiffness and cytoskeletal stability in FAK-/- cells is partially restored to wild-type level after rho-kinase inhibition with Y27632.
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| 3. |
- Klemm, Anna H, et al.
(författare)
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Comparing the mechanical influence of vinculin, focal adhesion kinase and p53 in mouse embryonic fibroblasts.
- 2009
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 379:3, s. 799-801
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Tidskriftsartikel (refereegranskat)abstract
- Cytoskeletal reorganization is an ongoing process when cells adhere, move or invade extracellular substrates. The cellular force generation and transmission are determined by the intactness of the actomyosin-(focal adhesion complex)-integrin connection. We investigated the intracellular course of action in mouse embryonic fibroblasts deficient in the focal adhesion proteins vinculin and focal adhesion kinase (FAK) and the nuclear matrix protein p53 using magnetic tweezer and nanoparticle tracking techniques. Results show that the lack of these proteins decrease cellular stiffness and affect cell rheological behavior. The decrease in cellular binding strength was higher in FAK- to vinculin-deficient cells, whilst p53-deficient cells showed no effect compared to wildtype cells. The intracellular cytoskeletal activity was lowest in wildtype cells, but increased in the following order when cells lacked FAK+p53>p53>vinculin. In summary, cell mechanical processes are differently affected by the focal adhesion proteins vinculin and FAK than by the nuclear matrix protein, p53.
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| 4. |
- Klemm, Anna H, et al.
(författare)
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Mechano-chemical signaling in F9 cells.
- 2006
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Ingår i: Cell Biology International. - : Wiley. - 1065-6995 .- 1095-8355. ; 30:9, s. 755-9
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Tidskriftsartikel (refereegranskat)abstract
- We investigated the molecular mechanism by which cells recognize and respond to physical forces in their local environment. Using a model system, to study wild type mouse F9 embryonic carcinoma cells, we examined how these cells sense mechanical stresses and translate them into biochemical responses through their cell surface receptor integrin and via the focal adhesion complex (FAC). Based on studies that show that many signal transducing molecules are immobilized on the cytoskeleton at the site of integrin binding within the focal adhesion complex, we found a time-dependent increase of focal adhesion kinase (pp125(FAK)) phosphorylation possibly due to protein kinase C (PKC) activation as well as protein kinase A (PKA) activity increase upon cell adhesion/spreading. These studies provide some insight into intracellular mechano-chemical signaling.
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| 5. |
- Klemm, Anna H, et al.
(författare)
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The influence of Pyk2 on the mechanical properties in fibroblasts.
- 2010
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 393:4, s. 694-7
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Tidskriftsartikel (refereegranskat)abstract
- The cell surface receptor integrin is involved in signaling mechanical stresses via the focal adhesion complex (FAC) into the cell. Within FAC, the focal adhesion kinase (FAK) and Pyk2 are believed to act as important scaffolding proteins. Based on the knowledge that many signal transducing molecules are transiently immobilized within FAC connecting the cytoskeleton with integrins, we applied magnetic tweezer and atomic force microscopic measurements to determine the influence of FAK and Pyk2 in cells mechanically. Using mouse embryonic fibroblasts (MEF; FAK(+/+), FAK(-/-), and siRNA-Pyk2 treated FAK(-/-) cells) provided a unique opportunity to describe the function of FAK and Pyk2 in more detail and to define their influence on FAC and actin distribution.
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| 6. |
- Raykova, Doroteya, 1986-, et al.
(författare)
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A method for Boolean analysis of protein interactions at a molecular level
- 2022
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Determination of interactions between native proteins in cells is important for understanding function. Here the authors report MolBoolean as a method to detect interactions between endogenous proteins in subcellular compartments, using antibody-DNA conjugates for identification and signal amplification. Determining the levels of protein-protein interactions is essential for the analysis of signaling within the cell, characterization of mutation effects, protein function and activation in health and disease, among others. Herein, we describe MolBoolean - a method to detect interactions between endogenous proteins in various subcellular compartments, utilizing antibody-DNA conjugates for identification and signal amplification. In contrast to proximity ligation assays, MolBoolean simultaneously indicates the relative abundances of protein A and B not interacting with each other, as well as the pool of A and B proteins that are proximal enough to be considered an AB complex. MolBoolean is applicable both in fixed cells and tissue sections. The specific and quantifiable data that the method generates provide opportunities for both diagnostic use and medical research.
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| 7. |
- Schewkunow, Vitali, et al.
(författare)
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Thermodynamic evidence of non-muscle myosin II-lipid-membrane interaction.
- 2008
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 366:2, s. 500-5
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Tidskriftsartikel (refereegranskat)abstract
- A unique feature of protein networks in living cells is that they can generate their own force. Proteins such as non-muscle myosin II are an integral part of the cytoskeleton and have the capacity to convert the energy of ATP hydrolysis into directional movement. Non-muscle myosin II can move actin filaments against each other, and depending on the orientation of the filaments and the way in which they are linked together, it can produce contraction, bending, extension, and stiffening. Our measurements with differential scanning calorimetry showed that non-muscle myosin II inserts into negatively charged phospholipid membranes. Using lipid vesicles made of DMPG/DMPC at a molar ratio of 1:1 at 10mg/ml in the presence of different non-muscle myosin II concentrations showed a variation of the main phase transition of the lipid vesicle at around 23 degrees C. With increasing concentrations of non-muscle myosin II the thermotropic properties of the lipid vesicle changed, which is indicative of protein-lipid interaction/insertion. We hypothesize that myosin tail binds to acidic phospholipids through an electrostatic interaction using the basic side groups of positive residues; the flexible, amphipathic helix then may partially penetrate into the bilayer to form an anchor. Using the stopped-flow method, we determined the binding affinity of non-muscle myosin II when anchored to lipid vesicles with actin, which was similar to a pure actin-non-muscle myosin II system. Insertion of myosin tail into the hydrophobic region of lipid membranes, a model known as the lever arm mechanism, might explain how its interaction with actin generates cellular movement.
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| 8. |
- Smith, James, et al.
(författare)
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CapZ-lipid membrane interactions : a computer analysis.
- 2006
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Ingår i: Theoretical Biology and Medical Modelling. - : Springer Science and Business Media LLC. - 1742-4682. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: CapZ is a calcium-insensitive and lipid-dependent actin filament capping protein, the main function of which is to regulate the assembly of the actin cytoskeleton. CapZ is associated with membranes in cells and it is generally assumed that this interaction is mediated by polyphosphoinositides (PPI) particularly PIP2, which has been characterized in vitro.RESULTS: We propose that non-PPI lipids also bind CapZ. Data from computer-aided sequence and structure analyses further suggest that CapZ could become partially buried in the lipid bilayer probably under mildly acidic conditions, in a manner that is not only dependent on the presence of PPIs. We show that lipid binding could involve a number of sites that are spread throughout the CapZ molecule i.e., alpha- and beta-subunits. However, a beta-subunit segment between residues 134-151 is most likely to be involved in interacting with and inserting into lipid membrane due to a slighly higher ratio of positively to negatively charged residues and also due to the presence of a small hydrophobic helix.CONCLUSION: CapZ may therefore play an essential role in providing a stable membrane anchor for actin filaments.
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| 9. |
- Beháňová, Andrea, et al.
(författare)
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Spatial Statistics for Understanding Tissue Organization
- 2022
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Ingår i: Frontiers in Physiology. - : Frontiers Media S.A.. - 1664-042X. ; 13
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Forskningsöversikt (refereegranskat)abstract
- Interpreting tissue architecture plays an important role in gaining a better understanding of healthy tissue development and disease. Novel molecular detection and imaging techniques make it possible to locate many different types of objects, such as cells and/or mRNAs, and map their location across the tissue space. In this review, we present several methods that provide quantification and statistical verification of observed patterns in the tissue architecture. We categorize these methods into three main groups: Spatial statistics on a single type of object, two types of objects, and multiple types of objects. We discuss the methods in relation to four hypotheses regarding the methods' capability to distinguish random and non-random distributions of objects across a tissue sample, and present a number of openly available tools where these methods are provided. We also discuss other spatial statistics methods compatible with other types of input data.
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| 10. |
- Cojoc, Gheorghe, et al.
(författare)
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Paired arrangement of kinetochores together with microtubule pivoting and dynamics drive kinetochore capture in meiosis I.
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Kinetochores are protein complexes on the chromosomes, whose function as linkers between spindle microtubules and chromosomes is crucial for proper cell division. The mechanisms that facilitate kinetochore capture by microtubules are still unclear. In the present study, we combine experiments and theory to explore the mechanisms of kinetochore capture at the onset of meiosis I in fission yeast. We show that kinetochores on homologous chromosomes move together, microtubules are dynamic and pivot around the spindle pole, and the average capture time is 3-4 minutes. Our theory describes paired kinetochores on homologous chromosomes as a single object, as well as angular movement of microtubules and their dynamics. For the experimentally measured parameters, the model reproduces the measured capture kinetics and shows that the paired configuration of kinetochores accelerates capture, whereas microtubule pivoting and dynamics have a smaller contribution. Kinetochore pairing may be a general feature that increases capture efficiency in meiotic cells.
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