Biology subjects

St Johnston, D.

Publications and source records attributed to St Johnston, D..

16 recordsLinked to original sources

bioRxiv2026

Fast single molecule localization microscopy in 3D

Super-resolution methods, such as single molecule localisation microscopy (SMLM), can determine the distribution of specific proteins at the nanometre scale. However, three-dimensional (3D) SMLM is typically limited to thin sections and requires prohibitively long acquisition times to image complete cells. Here we introduce a 3D SMLM imaging modality, Intrinsic3D, that combines the speed and optical sectioning of oblique plane microscopy (OPM) with the nanometre resolution of SMLM to determine the position of single molecule emitters throughout complete volumes more than an order-of-magnitude faster over regular DNA-PAINT. By scanning the oblique light sheet across the volume and combining localization information from two or more adjacent planes, an emitter's x, y and z position is determined, eliminating the need for post-processing steps to stitch multiple image stacks together. We demonstrate resolution and speed by imaging nuclear pore complexes in U2OS cells and Drosophila egg chambers. As a proof-of-concept, we map the 3D distribution of the cell-adhesion protein DE-cadherin in the Drosophila follicular epithelium and show that spot adherens junctions are enriched near tricellular junctions and contain more E-cadherin than the apical adherens junctions, suggesting that spot junctions play a wider role in cell-cell adhesion than previously appreciated. Finally, we examine the organization of Gliotactin, a tricellular septate junction protein relative to E-cadherin using exchange-PAINT. These investigations demonstrate that Intrinsic3D delivers rapid 3D protein mapping in whole cells. Combining Intrinsic3D with existing multiplex and resolution enhancing methods (e.g. exchange-PAINT and RESI) will enable multicolour, 3D in situ spatial proteomics in tissue samples and bridge the gap between SMLM and systems-level cell biology.

cell biology↗

bioRxiv2026

Integrin diversity enables dynamic adhesion control in a homeostatic epithelium

Homeostatic epithelia must balance stem cell maintenance, progenitor differentiation, and clearance of damaged cells while preserving barrier integrity. We investigated how integrin- ECM adhesion is regulated in the Drosophila midgut, a homeostatic epithelium with basal stem cells. The midgut expresses two beta integrins: ubiquitous {beta}Mys and endoderm-specific {beta}{nu}. ISCs and enteroblasts express only {beta}Mys, which pairs with Mew to mediate enteroblast attachment to the basement membrane. In contrast, enterocytes express both {beta}Mys and {beta}{nu}; Mew/{beta}{nu} supports ECM adhesion, while {beta}Mys pairs with Scab and localises to the basal labyrinth. Enterocytes lacking Mew or {beta}{nu} detach and are apically extruded, but this phenotype is rescued when the corresponding integrin is removed from the entire epithelium. Thus, enterocytes compete for basement membrane adhesion, with less adhesive cells being eliminated by their neighbours. In {beta}{nu} homozygotes, enteroblasts expand basally and adopt a migratory-like morphology. We propose that integrin-mediated competition for ECM adhesion is a general phenomenon that functions in the midgut to promote enterocyte extrusion, which stimulates the migration of nearby enteroblasts to maintain gut homeostasis.

developmental biology↗

bioRxiv2026

An aPKC rheostat induces apical contraction in response to epithelial stretching

Apical-basal polarity in epithelial cells is controlled by a conserved set of polarity factors that define the apical, junctional and basolateral domains of the cell, but how these factors adapt to or control changes in domain sizes during cell shape changes remains unclear. Atypical protein kinase C (aPKC) is the main effector of apical identity, phosphorylating the lateral factors, Bazooka/Par-3, Lgl, Par-1 and Yurt to exclude them from the apical domain. Using analogue-sensitive aPKC in Drosophila follicle cells, we found that aPKC substrates differ over 100-fold in their sensitivity to inhibition, revealing a hierarchy of substrates, that is conserved in mammals, in which high-affinity substrates out-compete low-affinity substrates when aPKC activity is limiting. Mild aPKC inhibition prevents the phosphorylation of its lowest affinity substrate, Yurt. Yurt then accumulates apically by binding to Crumbs, where it activates apical constriction through Shroom, Cysts/Dp114RhoGEF, Rho kinase and Myosin. Yurt localises apically in cells that are stretched, either by morphogenesis or artificially, indicating that stretching reduces aPKC activity to trigger an antagonistic contraction. By contrast, yurt- cells fail to resist stretching. Thus, the aPKC/Yurt pathway functions as a homeostatic stretch response, in which apical and lateral epithelial polarity factors collaborate to mechanically regulate apical domain size.

cell biology↗

bioRxiv2024

Adherens junctions limit the extent of the septate junctions in Drosophila midgut enterocytes but are not required for apical-basal polarity

Adherens junctions formed by E-cadherin adhesion complexes play central roles in the organisation and apical-basal polarisation of both mammalian and insect epithelia. Here we investigate the function of the components of the E-cadherin adhesion complex in the Drosophila midgut epithelium, which establishes polarity by a different mechanism from other fly epithelia and has an inverted junctional arrangement, in which the adherens junctions lie below the septate junctions. Unlike other epithelial tissues, loss of E-cadherin, Armadillo ({beta}-catenin) or -catenin has no effect on the polarity or organisation of the adult midgut epithelium. This is not due to redundancy with N-cadherin, providing further evidence that the midgut polarises in distinct way from other epithelia. However, E-cadherin (shg) and armadillo mutants have an expanded septate junction domain and a smaller lateral domain below the septate junctions. Thus, E-cadherin adhesion complexes limit the basal extent of the septate junctions. This function does not appear to depend on the linkage of E-cadherin to the actin cytoskeleton because -catenin mutants do not significantly perturb the relative sizes of the septate and sub-septate junction domains.

cell biology↗

bioRxiv2024

Tracking exocytic vesicle movements with MSP-tracker/viewer reveals the spatial control of secretion in epithelial cells.

Understanding how specific secretory cargoes are targeted to distinct domains of the plasma membrane in epithelial cells requires analysing the trafficking of post-Golgi vesicles to their sites of secretion. We used the RUSH (retention using selective hooks) system to synchronously release an apical cargo, Cadherin 99C (Cad99C), and a basolateral cargo, the ECM protein Nidogen, from the endoplasmic reticulum and follow their movements to the plasma membrane. We also developed an interactive vesicle tracking framework, MSP-tracker and viewer, that exploits developments in computer vision and deep learning to determine vesicle trajectories in a noisy environment without the need for extensive training data. MSP-tracker outperformed other tracking software in detecting and tracking post-Golgi vesicles, revealing that Cad99c vesicles predominantly move apically with a mean speed of 1.1{micro}m/sec. This is reduced to 0.85 {micro}m/sec by a dominant slow dynein mutant, demonstrating that dynein transports Cad99C vesicles to the apical cortex. Furthermore, both the dynein mutant and microtubule depolymerisation cause lateral Cad99C secretion. Thus, microtubule organisation plays a central role in targeting apical secretion, suggesting that Drosophila does not have distinct apical versus basolateral vesicle fusion machinery. Nidogen vesicles undergo planar-polarised transport to the leading edge of follicle cells as they migrate over the ECM, whereas most Collagen is secreted at trailing edges. The follicle cells therefore bias secretion of different ECM components to opposite sides of the cell, revealing that the secretory pathway is more spatially organised than previously thought.

cell biology↗

bioRxiv2024

User-friendly Oblique Plane Microscopy on a fully functional commercially available microscope base

In this work we present an Oblique Plane Microscope designed to work seamlessly with a commercially available microscope base. To support all the functionality offered by the microscope base, where the position of the objective lens is not fixed, we adopted a two-mirror scanning geometry that can compensate for changes to the position of the objective lens during routine microscope operation. We showed that within the expected displacement range of the 100X, 1.35 NA objective lens away from its designed position, and for most practical applications, there is no significant effect on the resolving power, or the fidelity of the 3D data produced by the microscope. Compared to the more traditional scan-lens/galvo-mirror combination, the two-mirror scanning geometry offers higher light-efficiency and a more compact footprint, which could be beneficial to all OPM designs regardless of the use of a commercial base or not.

biophysics↗

bioRxiv2023

The Shot CH1 domain recognises a distinct form of F-actin during Drosophila oocyte determination

As in mammals, only one cell in a Drosophila multicellular female germline cyst is specified as an oocyte. The symmetry-breaking cue for oocyte selection is provided by the fusome, a tubular structure connecting all cells in the cyst. The Drosophila spectraplakin Shot localises to the fusome and translates its asymmetry into a polarised microtubule network that is essential for oocyte specification, but how Shot recognises the fusome is unclear. Here we demonstrate that Shots actin-binding domain (ABD) is necessary and sufficient to localise Shot to the fusome and mediates Shot function in oocyte specification together with the microtubule-binding domains. The calponin homology domain 1 (CH1) of Shots ABD recognises fusomal F-actin and requires CH2 to distinguish it from other forms of F-actin in the cyst. By contrast, the ABDs of Utrophin, Fimbrin, Filamin, Lifeact and F-tractin do not recognise fusomal F-actin. We therefore propose that Shot propagates fusome asymmetry by recognising a specific conformational state of F-actin on the fusome.

cell biology↗

bioRxiv2021

De novo apical domain formation inside the Drosophila adult midgut epithelium

In the adult Drosophila midgut, basal intestinal stem cells give rise to enteroblasts that integrate into the epithelium as they differentiate into enterocytes. Integrating enteroblasts must generate a new apical domain and break through the septate junctions between neighbouring enterocytes, while maintaining barrier function. We observe that enteroblasts form an apical membrane initiation site when they reach the septate junction between the overlying enterocytes. A space appears above the enteroblast as Cadherin clears from its apical surface. New septate junctions then form laterally with neighbouring enterocytes and the AMIS develops into pre-apical compartment with a fully-formed brush border. Finally, the overlying septate junction dissolves and the pre-enterocyte reaches the gut lumen. Enteroblasts therefore form an apical domain before they have a free apical surface. The process of enteroblast integration resembles lumen formation in mammalian epithelial cysts, highlighting the similarities between the fly midgut and mammalian epithelia.

cell biology↗

bioRxiv2021

A method for single molecule localization microscopy of tissues reveals nonrandom distribution of nuclear pores in Drosophila

Single Molecule Localisation Microscopy (SMLM) can provide nanoscale resolution in thin samples but has rarely been applied to tissues, because of high background from out of focus emitters. Here we describe a line scanning microscope that provides optical sectioning for SMLM in tissues. Imaging endogenously-tagged nucleoporins and F-actin on this system using DNA- and peptide-PAINT routinely gives 30nm resolution or better at depths greater than 20 {micro}m. This revealed that the nuclear pores are nonrandomly distributed in most Drosophila tissues, in contrast to cultured cells. Lamin Dm0 shows a complementary localisation to the nuclear pores, suggesting that it corrals the pores. Furthermore, ectopic expression of the tissue-specific Lamin C distributes the nuclear pores more randomly, whereas lamin C mutants enhance nuclear pore clustering, particularly in muscle nuclei. Since nucleoporins interact with specific chromatin domains, nuclear pore clustering could regulate chromatin organisation locally and contribute to the disease phenotypes caused by human Lamin A/C laminopathies.

cell biology↗

bioRxiv2021

Symmetry breaking in the female germline cyst

In mammals and flies, only a limited number of cells in a multicellular female germline cyst become oocytes, but how the oocyte is selected is unknown. Here we show that the microtubule minus end-stabilizing protein, Patronin/CAMSAP marks the future Drosophila oocyte and is required for oocyte specification. The spectraplakin, Shot, recruits Patronin to the fusome, a branched structure extending into all cyst cells. Patronin stabilizes more microtubules in the cell with most fusome and this weak asymmetry is amplified by Dynein-dependent transport of Patronin-stabilized microtubules. This forms a polarized microtubule network, along which Dynein transports oocyte determinants into the presumptive oocyte. Thus, Patronin amplifies a weak fusome anisotropy to break cyst symmetry. These findings reveal a molecular mechanism of oocyte selection in the germline cyst.

developmental biology↗

bioRxiv2021

The Drosophila anterior-posterior axis is polarized by asymmetric myosin activation

The Drosophila anterior-posterior (AP) axis is specified at mid-oogenesis when Par-1 kinase is recruited to the posterior cortex of the oocyte, where it polarises the microtubule cytoskeleton to define where the axis determinants, bicoid and oskar mRNAs localise. This polarity is established in response to an unknown signal from the follicle cells, but how this occurs is unclear. Here we show that the myosin chaperone, Unc-45 and Non-Muscle Myosin II (MyoII) are required in the germ line upstream of Par-1 in polarity establishment. Furthermore, the Myosin regulatory Light Chain (MRLC) is di-phosphorylated at the oocyte posterior in response to the follicle cell signal, inducing longer pulses of myosin contractility at the posterior and increased cortical tension. Over-expression of MRLC-T21A that cannot be di-phosphorylated or acute treatment with the Myosin light chain kinase inhibitor ML-7 abolish Par-1 localisation, indicating that posterior of MRLC di-phosphorylation is essential for polarity. Thus, asymmetric myosin activation polarizes the anterior-posterior axis by recruiting and maintaining Par-1 at the posterior cortex. This raises an intriguing parallel with AP axis formation in C. elegans where MyoII is also required to establish polarity, but functions to localize the anterior PAR proteins rather than PAR-1.

developmental biology↗

bioRxiv2020

RhoGAP19D inhibits Cdc42 laterally to control epithelial cell shape and prevent invasion

Cdc42-GTP is required for apical domain formation in epithelial cells where it recruits and activates the Par-6/aPKC polarity complex, but how the activity of Cdc42 itself is restricted apically unclear. We used sequence analysis and 3D structure modelling to determine which Drosophila GTPase Activating Proteins (GAPs) are likely to interact with Cdc42 and identified RhoGAP19D as the sole Cdc42GAP required for polarity in the follicle cell epithelium. RhoGAP19D is recruited by -catenin to lateral E-cadherin adhesion complexes, resulting in exclusion of active Cdc42 from the lateral domain. rhogap19d mutants therefore lead to lateral Cdc42 activity, which expands the apical domain through increased Par-6/aPKC activity and stimulates lateral contractility through the myosin light chain kinase, Genghis khan (MRCK). This causes buckling of the epithelium and invasion into the adjacent tissue, a phenotype resembling that of pre-cancerous breast lesions. Thus, RhoGAP19D couples lateral Cadherin adhesion to the apical localisation of active Cdc42, thereby suppressing epithelial invasion.

cell biology↗

bioRxiv2019

The role of integrins in Drosophila egg chamber morphogenesis

A Drosophila egg chamber is comprised of a germline cyst surrounded by a tightly-organised epithelial monolayer, the follicular epithelium (FE). Loss of integrin function from the FE disrupts epithelial organisation at egg chamber termini, but the cause of this phenotype remains unclear. Here we show that the {beta}-integrin Myospheroid (Mys) is only required during early oogenesis when the pre-follicle cells form the FE. mys mutants disrupt both the formation of a monolayered epithelium at egg chamber termini and the morphogenesis of the stalk between adjacent egg chambers, which develops through the intercalation of two rows of cells into a single-cell wide stalk. Secondary epithelia, like the FE, have been proposed to require adhesion to the basement membrane to polarise. However, Mys is not required for pre-follicle cell polarisation, as both follicle and stalk cells localise polarity factors correctly, despite being mispositioned. Instead, loss of integrins causes pre-follicle cells to basally constrict, detach from the basement membrane and become internalised. Thus, integrin function is dispensable for pre-follicle cell polarity but is required to maintain cellular organisation and cell shape during morphogenesis.

developmental biology↗

bioRxiv2018

Drosophila IMP regulates Kuzbanian to control the timing of Notch signalling in the follicle cells

The timing of Drosophila egg chamber development is controlled by a germline Delta signal that activates Notch in the follicle cells to induce them to cease proliferation and differentiate. Here we report that follicle cells lacking the RNA-binding protein IMP go through one extra division due to a delay in the Delta-dependent S2 cleavage of Notch. The timing of Notch activation has previously been shown to be controlled by cis-inhibition by Delta in the follicle cells, which is relieved when the miRNA pathway represses Delta expression. imp mutants are epistatic to Delta mutants and give an additive phenotype with belle and dicer mutants, indicating that IMP functions independently of both cis-inhibition and the miRNA pathway. We find that the imp phenotype is rescued by over-expression of Kuzbanian, the metalloprotease that mediates the Notch S2 cleavage. Furthermore, Kuzbanian is not enriched at the apical membrane in imp mutants, accumulating instead in late endosomes. Thus, IMP regulates Notch signalling by controlling the localisation of Kuzbanian to the apical domain, where Notch cleavage occurs, revealing a novel regulatory step in the Notch pathway.\n\nSummaryIMP regulates Notch signalling in follicle cells by controlling Kuzbanian localisation to the apical domain, where Notch cleavage occurs, revealing a novel regulatory step in the Notch pathway.

developmental biology↗

bioRxiv2018

An alternative mode of epithelial polarity in the Drosophila midgut

Apical-basal polarity is essential for the formation and function of epithelial tissues, whereas loss of polarity is a hallmark of tumours. Studies in Drosophila have identified conserved polarity factors that define the apical (Crumbs, Stardust, Par-6, aPKC), junctional (Baz/Par-3) and basolateral (Scribbled, Discs large, Lgl) domains of epithelial cells (1, 2). Because these conserved factors mark equivalent domains in diverse vertebrate and invertebrate epithelial types, it is generally assumed that this system organises polarity in all epithelia. Here we show that this is not the case, as none of these canonical factors are required for the polarisation of the endodermal epithelium of the Drosophila adult midgut. Furthermore, unlike other Drosophila epithelia, the midgut forms occluding junctions above adherens junctions, as in vertebrates, and requires the integrin adhesion complex for polarity (3, 4). Thus, Drosophila contains two types of epithelia that polarise by different mechanisms. Since knock-outs of canonical polarity factors often have little effect on the polarity of vertebrate epithelia, this diversity of polarity mechanisms is likely to be conserved in other animals (5-8).

cell biology↗

bioRxiv2017

Localised Dynactin Protects Growing Microtubules To Deliver Oskar mRNA To The Posterior Cortex Of The Drosophila Oocyte

The localisation of oskar mRNA to the posterior of the Drosophila oocyte defines where the abdomen and germ cells form in the embryo. Kinesin 1 transports oskar mRNA to the oocyte posterior along a polarised microtubule cytoskeleton that grows from non-centrosomal microtubule organising centres (ncMTOCs) along the anterior/lateral cortex. Here we show that the formation of this polarised microtubule network also requires the posterior regulation of microtubule growth. A mutation in the Dynactin Arp1 subunit causes most oskar mRNA to localise in the posterior cytoplasm rather than cortically. oskar mRNA transport and anchoring are normal in this mutant, but the microtubules fail to reach the posterior pole. Thus, Dynactin acts as an anti-catastrophe factor that extends microtubule growth posteriorly. Kinesin 1 transports Dynactin to the oocyte posterior, creating a positive feedback loop that increases the length and persistence of the posterior microtubules that deliver oskar mRNA to the cortex.

cell biology↗