Biology subjects

Troughton, L. D.

Publications and source records attributed to Troughton, L. D..

7 recordsLinked to original sources

bioRxiv2023

Non-muscle myosin 2 filaments are processive in cells

Directed transport of cellular components is often dependent on the processive movements of cytoskeletal motors. Myosin 2 motors predominantly engage actin filaments of opposing orientation to drive contractile events, and are therefore not traditionally viewed as processive. However, recent in vitro experiments with purified non-muscle myosin 2 (NM2) demonstrated myosin 2 filaments could move processively. Here, we establish processivity as a cellular property of NM2. Processive runs in central nervous system-derived CAD cells are most apparent as processive movements on bundled actin in protrusions that terminate at the leading edge. We find that processive velocities in vivo are consistent with in vitro measurements. NM2 makes these processive runs in its filamentous form against lamellipodia retrograde flow, though anterograde movement can still occur in the absence of actin dynamics. Comparing the processivity of NM2 isoforms, we find that NM2A moves slightly faster than NM2B. Finally, we demonstrate that this is not a cell-specific property, as we observe processive-like movements of NM2 in the lamella and subnuclear stress fibers of fibroblasts. Collectively, these observations further broaden NM2 functionality and the biological processes in which the already ubiquitous motor can contribute.

cell biology↗

bioRxiv2022

Atypical peripheral actin band formation via overactivation of RhoA and Non-muscle myosin II in Mitofusin 2 deficient cells

Cell spreading and migration play central roles in many physiological and pathophysiological processes. We have previously shown that MFN2 regulates the migration of human neutrophillike cells via suppressing Rac activation. Here, we show that in mouse embryonic fibroblasts, MFN2 suppresses RhoA activation and supports cell polarization. After the initial spreading period, the wild-type cells polarize and migrate, whereas the Mfn2-/- cells maintain a circular shape. Increased cytosolic Ca2+ resulting from the loss of Mfn2 is directly responsible for this phenotype, which can be rescued by expressing an artificial tether to bring mitochondria and ER to close vicinity. Elevated cytosolic Ca2+ activates Ca2+/calmodulin-dependent protein kinase II, RhoA, and Myosin light-chain kinase, causing an over-activation of non-muscle myosin II and a formation of a prominent F-actin ring at the cell periphery and increased cell contractility. The formation of the peripheral actin band alters cell physics and is dependent on substrate rigidity. Our results provide a novel molecular basis to understand how MFN2 regulates distinct signaling pathways in different cells and tissue environments, which is instrumental in understanding and treating MFN2-related diseases.

cell biology↗

bioRxiv2021

A Workflow for Rapid Unbiased Quantification of Fibrillar Feature Alignment in Biological Images

Measuring the organisation of the cellular cytoskeleton and the surrounding extracellular matrix (ECM) is currently of wide interest as changes in both local and global alignment can highlight alterations in cellular functions and material properties of the extracellular environment. Different approaches have been developed to quantify these structures, typically based on fibre segmentation or on matrix representation and transformation of the image, each with its own advantages and disadvantages. Here we present AFT - Alignment by Fourier Transform, a workflow to quantify the alignment of fibrillar features in microscopy images exploiting 2D Fast Fourier Transforms (FFT). Using pre-existing datasets of cell and ECM images, we demonstrate our approach and compare and contrast this workflow with two other well-known ImageJ algorithms to quantify image feature alignment. These comparisons reveal that AFT has a number of advantages due to its grid-based FFT approach. 1) Flexibility in defining the window and neighbourhood sizes allows for performing a parameter search to determine an optimal length scale to carry out alignment metrics. This approach can thus easily accommodate different image resolutions and biological systems. 2) The length scale of decay in alignment can be extracted by comparing neighbourhood sizes, revealing the overall distance that features remain anisotropic. 3) The approach is ambivalent to the signal source, thus making it applicable for a wide range of imaging modalities and is dependent on fewer input parameters than segmentation methods. 4) Finally, compared to segmentation methods, this algorithm is computationally inexpensive, as high-resolution images can be evaluated in less than a second on a standard desktop computer. This makes it feasible to screen numerous experimental perturbations or examine large images over long length scales. Implementation is made available in both MATLAB and Python for wider accessibility, with example datasets for single images and batch processing. Additionally, we include an approach to automatically search parameters for optimum window and neighbourhood sizes, as well as to measure the decay in alignment over progressively increasing length scales.

biophysics↗

bioRxiv2020

Laminin N-terminus α31 expression during development in an inducible-transgenic mouse model is lethal and causes a multitude of tissue-specific defects

Laminins are essential components of all basement membranes where they regulate an extensive array of tissue functions. Alternative splicing from the laminin 3 gene produces a non-laminin but netrin-like protein, Laminin N terminus 31 (LaNt 31). LaNt 31 is widely expressed in intact tissue and is upregulated in epithelial cancers and during wound healing. In vitro functional studies have shown that LaNt 31 can influence numerous aspects of epithelial cell behaviour via modifying matrix organisation, suggesting a new model of laminin auto-regulation. However, the function of this protein has not been established beyond the epithelium and it has never been studied in vivo. Here, a mouse transgenic line was generated using the ubiquitin C promoter to drive inducible expression of LaNt 31. When expression was induced at embryonic day 15.5, LaNt 31 transgenic animals were not viable at birth, exhibiting localised regions of erythema. Numerous striking defects were apparent histologically, including extra-vascular erythrocytes in multiple tissues, kidney epithelial detachment, tubular dilation, interstitial bleeding, and thickening of tubule basement membranes, disruption of the epidermal basal cell layer and of the hair follicle outer root sheath, and ~50% reduction of cell numbers in the liver associated with depletion of hematopoietic erythrocytic foci. These findings demonstrate that LaNt 31 can influence tissue morphogenesis during development. More broadly, these data provide the first in vivo evidence to support an emerging model of laminin self-regulation and provide a valuable model for onward investigation into this important area. Summary StatementExpression during development of Laminin N-terminus 31, a netrin-like laminin splice isoform, caused defects indicating basement membrane disruption in multiple tissues; providing the first in vivo evidence for laminin self-regulation.

cell biology↗

bioRxiv2020

Laminin N-terminus α31 is upregulated in invasive ductal breast cancer and changes the mode of tumour invasion.

Laminin N-terminus 31 (LaNt 31) is an alternative splice isoform derived from the laminin 3 gene. The LaNt 31 protein is enriched around the terminal duct lobular units in normal breast tissue. In the skin and cornea the protein influences epithelial cell migration and tissue remodelling. However, LaNt 31 has never been investigated in a tumour environment. Here we analysed LaNt 31 in invasive ductal carcinoma and determined its contribution to breast carcinoma invasion. LaNt 31 expression and distribution were analysed by immunohistochemistry in human breast tissue biopsy sections and tissue microarrays covering 232 breast cancer samples. This analysis revealed LaNt 31 to be upregulated in 56 % of invasive ductal carcinoma specimens compared with matched normal tissue, and further increased in nodal metastasis compared with the tumour mass in 45 % of samples. 65.8 % of triple negative cases displayed medium to high LaNt 31 expression. To study LaNt 31 function, an adenoviral system was used to induce expression in MCF-7 and MDA-MB-231 cells. Metabolic activity, 2D cell migration, and invasion into collagen hydrogels were not significantly different between LaNt 31 overexpressing cells and control treated cells. However, LaNt 31 overexpressing MDA-MB-231 cells displayed a striking change in their mode of invasion into laminin-containing Matrigel; changing from multicellular streaming to individual cellular-invasion. In agreement with these results, 66.7% of the tumours with the highest LaNt 31 expression were non-cohesive. Together these findings indicate that breast cancer-associated changes in LaNt 31 expression could directly contribute to tumour invasiveness, and that this little-studied protein may become a therapeutic target.

cancer biology↗

bioRxiv2020

Laminin N-terminus α31 protein distribution in adult human tissues

Laminin N terminus 31 (LaNt 31) is a netrin-like protein derived from alternative splicing of the laminin 3 gene. Although LaNt 31 has been demonstrated to influence corneal and skin epithelial cell function, its expression has not been investigated beyond these tissues. In this study, we used immunohistochemistry to characterise the distribution of this protein in a wide-array of human tissue sections in comparison to laminin 3. These data revealed widespread LaNt 31 expression. In epithelial tissue, LaNt 31 was present in the basal layer of the epidermis, throughout the epithelium of the digestive tract, and much of the epithelium of the reproductive system. LaNt 31 was also found throughout the vasculature of most tissues, with enrichment in reticular-like fibres in the extracellular matrix surrounding large vessels. A similar matrix pattern was observed around the terminal ducts in the breast and around the alveolar epithelium in the lung, where basement membrane staining was also evident. Specific enrichment of LaNt 31 was identified in sub-populations of cells of the kidney, liver, pancreas, and spleen, with variations in intensity between different cell types in the collecting ducts and glomeruli of the kidney being of particular note. Intriguingly, LaNt 31 immunoreactivity was also evident in neurons of the central nervous system, in the cerebellum, cerebral cortex, and spinal cord. Together these findings suggest that LaNt 31 may be functionally relevant in a wider range of tissue contexts than previously thought, and provides a valuable basis for investigation into this interesting protein.

cell biology↗

bioRxiv2019

LaNt α31 modulates LM332 organisation during matrix deposition leading to cell-matrix adhesion and migration defects.

Laminin N-terminus 31 (LaNt 31), a member of the laminin superfamily, expressed at low levels in intact epithelium but upregulated during wound repair. Increased expression of LaNt 31 reduced migration rate of corneal keratinocytes through an unknown mechanism. Here, we investigated whether LaNt 31 influences cell behaviour through modulating laminin-mediated processes. Adenoviral delivery of LaNt 31 into corneal epithelial cells led to reduced migration speed and increased cell spreading and changed laminin 332 organisation from diffuse arcs to tight clusters. Enhanced recruitment of collagen XVII and bullous pemphigoid antigen 1e to {beta}4 integrin, indicating early maturation of hemidesmosomes, and changed focal adhesion distribution were also identified. LaNt 31 and laminin {beta}3 co-immunoprecipitated from doubly transduced cells and were deposited together in live imaging experiment. Moreover, LaNt 31 expression led to increased matrix metalloproteinase (MMP) activity and proteolytic processing of laminin 3, and the inhibition of MMP activity rescued the laminin and hemidesmosome phenotypes. Provision of cell-derived extracellular matrix rescued the cell spreading and motility effects. These findings reveal LaNt 31 as a new player in regulating cell-to-matrix adhesion through its ability to influence laminin organisation and proteolytic processing.

cell biology↗