The crucial role of bioimage analysts in scientific research and publication.

Bioimage analysis (BIA), a crucial discipline in biological research, overcomes the limitations of subjective analysis in microscopy through the creation and application of quantitative and reproducible methods. The establishment of dedicated BIA support within academic institutions is vital to improving research quality and efficiency and can significantly advance scientific discovery. However, a lack of training resources, limited career paths and insufficient recognition of the contributions made by bioimage analysts prevent the full realization of this potential. This Perspective - the result of the recent The Company of Biologists Workshop 'Effectively Communicating Bioimage Analysis', which aimed to summarize the global BIA landscape, categorize obstacles and offer possible solutions - proposes strategies to bring about a cultural shift towards recognizing the value of BIA by standardizing tools, improving training and encouraging formal credit for contributions. We also advocate for increased funding, standardized practices and enhanced collaboration, and we conclude with a call to action for all stakeholders to join efforts in advancing BIA.

Liquid-liquid phase separation of type II diabetes-associated IAPP initiates hydrogelation and aggregation.

Amyloidoses (misfolded polypeptide accumulation) are among the most debilitating diseases our aging societies face. Amyloidogenesis can be catalyzed by hydrophobic-hydrophilic interfaces (e.g., air-water interface in vitro [AWI]). We recently demonstrated hydrogelation of the amyloidogenic type II diabetes-associated islet amyloid polypeptide (IAPP), a hydrophobic-hydrophilic interface-dependent process with complex kinetics. We demonstrate that human IAPP undergoes AWI-catalyzed liquid-liquid phase separation (LLPS), which initiates hydrogelation and aggregation. Insulin modulates these processes but does not prevent them. Using nonamyloidogenic rat IAPP, we show that, whereas LLPS does not require the amyloidogenic sequence, hydrogelation and aggregation do. Interestingly, both insulin and rat sequence delayed IAPP LLPS, which may reflect physiology. By developing an experimental setup and analysis tools, we show that, within the whole system (beyond the droplet stage), macroscopic interconnected aggregate clusters form, grow, fuse, and evolve via internal rearrangement, leading to overall hydrogelation. As the AWI-adsorbed gelled layer matures, its microviscosity increases. LLPS-driven aggregation may be a common amyloid feature and integral to pathology.

The kinetics of islet amyloid polypeptide phase-separated system and hydrogel formation are critically influenced by macromolecular crowding.

Many protein misfolding diseases (e.g. type II diabetes and Alzheimer's disease) are characterised by amyloid deposition. Human islet amyloid polypeptide (hIAPP, involved in type II diabetes) spontaneously undergoes liquid-liquid phase separation (LLPS) and a kinetically complex hydrogelation, both catalysed by hydrophobic-hydrophilic interfaces (e.g. air-water interface and/or phospholipids-water interfaces). Gelation of hIAPP phase-separated liquid droplets initiates amyloid aggregation and the formation of clusters of interconnected aggregates, which grow and fuse to eventually percolate the whole system. Droplet maturation into irreversible hydrogels via amyloid aggregation is thought to be behind the pathology of several diseases. Biological fluids contain a high volume fraction of macromolecules, leading to macromolecular crowding. Despite crowding agent addition in in vitro studies playing a significant role in changing protein phase diagrams, the mechanism underlying enhanced LLPS, and the effect(s) on stages beyond LLPS remain poorly or not characterised.We investigated the effect of macromolecular crowding and increased viscosity on the kinetics of hIAPP hydrogelation using rheology and the evolution of the system beyond LLPS by microscopy. We demonstrate that increased viscosity exacerbated the kinetic variability of hydrogelation and of the phase separated-aggregated system, whereas macromolecular crowding abolished heterogeneity. Increased viscosity also strengthened the gel meshwork and accelerated aggregate cluster fusion. In contrast, crowding either delayed cluster fusion onset (dextran) or promoted it (Ficoll). Our study highlights that an in vivo crowded environment would critically influence amyloid stages beyond LLPS and pathogenesis.

Nucleoplasmic Reticulum Formation in Human Endometrial Cells is Steroid Hormone Responsive and Recruits Nascent Components.

The nuclei of cells may exhibit invaginations of the nuclear envelope under a variety of conditions. These invaginations form a branched network termed the nucleoplasmic reticulum (NR), which may be found in cells in pathological and physiological conditions. While an extensive NR is a hallmark of cellular senescence and shows associations with some cancers, very little is known about the formation of NR in physiological conditions, despite the presence of extensive nuclear invaginations in some cell types such as endometrial cells. Here we show that in these cells the NR is formed in response to reproductive hormones. We demonstrate that oestrogen and progesterone are sufficient to induce NR formation and that this process is reversible without cell division upon removal of the hormonal stimulus. Nascent lamins and phospholipids are incorporated into the invaginations suggesting that there is a dedicated machinery for its formation. The induction of NR in endometrial cells offers a new model to study NR formation and function in physiological conditions.

Sister chromatid cohesion is mediated by individual cohesin complexes.

Eukaryotic genomes are organized by loop extrusion and sister chromatid cohesion, both mediated by the multimeric cohesin protein complex. Understanding how cohesin holds sister DNAs together, and how loss of cohesion causes age-related infertility in females, requires knowledge as to cohesin's stoichiometry in vivo. Using quantitative super-resolution imaging, we identified two discrete populations of chromatin-bound cohesin in postreplicative human cells. Whereas most complexes appear dimeric, cohesin that localized to sites of sister chromatid cohesion and associated with sororin was exclusively monomeric. The monomeric stoichiometry of sororin:cohesin complexes demonstrates that sister chromatid cohesion is conferred by individual cohesin rings, a key prediction of the proposal that cohesion arises from the co-entrapment of sister DNAs.

The enigma of phosphoinositides and their derivatives: Their role in regulation of subcellular compartment morphology.

The general segregation of a molecular class, lipids, from the pathways of cellular communication, via endo-membranes, has resulted in the over-simplification and misconceptions in deciphering cell signalling mechanisms. Mechanisms in signal transduction and protein activation require targeting of proteins to membranous compartments with a specific localised morphology and dynamics that are dependent on their lipid composition. Many posttranslational events define cellular behaviours and without the active role of membranous compartments these events lead to various dysregulations of the signalling pathways. We summarise the key findings, using tools such as the rapalogue dimerisation, in the structural roles and signalling of the inter-related phosphoinositide lipids and their derivative, diacylglycerol, in the regulation of nuclear envelope biogenesis and other subcellular compartments such as the nucleoplasmic reticulum.

Host Vesicle Fusion Protein VAPB Contributes to the Nuclear Egress Stage of Herpes Simplex Virus Type-1 (HSV-1) Replication.

The primary envelopment/de-envelopment of Herpes viruses during nuclear exit is poorly understood. In Herpes simplex virus type-1 (HSV-1), proteins pUL31 and pUL34 are critical, while pUS3 and some others contribute; however, efficient membrane fusion may require additional host proteins. We postulated that vesicle fusion proteins present in the nuclear envelope might facilitate primary envelopment and/or de-envelopment fusion with the outer nuclear membrane. Indeed, a subpopulation of vesicle-associated membrane protein-associated protein B (VAPB), a known vesicle trafficking protein, was present in the nuclear membrane co-locating with pUL34. VAPB knockdown significantly reduced both cell-associated and supernatant virus titers. Moreover, VAPB depletion reduced cytoplasmic accumulation of virus particles and increased levels of nuclear encapsidated viral DNA. These results suggest that VAPB is an important player in the exit of primary enveloped HSV-1 virions from the nucleus. Importantly, VAPB knockdown did not alter pUL34, calnexin or GM-130 localization during infection, arguing against an indirect effect of VAPB on cellular vesicles and trafficking. Immunogold-labelling electron microscopy confirmed VAPB presence in nuclear membranes and moreover associated with primary enveloped HSV-1 particles. These data suggest that VAPB could be a cellular component of a complex that facilitates UL31/UL34/US3-mediated HSV-1 nuclear egress.

Formation of a nucleoplasmic reticulum requires de novo assembly of nascent phospholipids and shows preferential incorporation of nascent lamins.

Structure of interphase cell nuclei remains dynamic and can undergo various changes of shape and organisation, in health and disease. The double-membraned envelope that separates nuclear genetic material from the rest of the cell frequently includes deep, branching tubular invaginations that form a dynamic nucleoplasmic reticulum (NR). This study addresses mechanisms by which NR can form in interphase nuclei. We present a combination of Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS) approach and light microscopy techniques to follow formation of NR by using pulse-chase experiments to examine protein and lipid delivery to nascent NR in cultured cells. Lamina protein incorporation was assessed using precursor accumulation (for lamin A) or a MAPLE3 photoconvertible tag (for lamin B1) and membrane phospholipid incorporation using stable isotope labelling with deuterated precursors followed by high resolution NanoSIMS. In all three cases, nascent molecules were selectively incorporated into newly forming NR tubules; thus strongly suggesting that NR formation is a regulated process involving a focal assembly machine, rather than simple physical perturbation of a pre-existing nuclear envelope.

Stress-induced release of Oct-1 from the nuclear envelope is mediated by JNK phosphorylation of lamin B1.

The nuclear lamina can bind and sequester transcription factors (TFs), a function lost if the lamina is abnormal, with missing or mutant lamin proteins. We now show that TF sequestration is not all-or-nothing, but a dynamic physiological response to external signals. We show that the binding of the ubiquitous TF, Oct-1, to lamin B1 was reversed under conditions of cellular stress caused, inter alia, by the chemical methylating agent methylmethanesulfonate (MMS). A search for lamin B1 post-translational modifications that might mediate changes in Oct-1 binding using kinase inhibitors uncovered a role for c-Jun N-terminal kinase (JNK). Phosphoproteomic and site-directed mutagenesis analyses of lamin B1 isolated from control and MMS-treated nuclei identified T575 as a JNK site phosphorylated after stress. A new phospho-T575 specific anti-peptide antibody confirmed increased interphase cellular T575 phosphorylation after cell exposure to certain stress conditions, enabling us to conclude that lamin B1 acts as an interphase kinase target, releasing Oct-1 to execute a protective response to stress.

Zeaxanthin from Porphyridium purpureum induces apoptosis in human melanoma cells expressing the oncogenic BRAF V600E mutation and sensitizes them to the BRAF inhibitor vemurafenib

Abstract Zeaxanthin, an abundant carotenoid present in fruits, vegetables and algae was reported to exert antiproliferative activity and induce apoptosis in human uveal melanoma cells. It also inhibited uveal melanoma tumor growth and cell migration in nude mice xenograft models. Here we report that zeaxanthin purified from the rhodophyte Porphyridium purpureum (Bory) K.M.Drew & R.Ross, Porphyridiaceae, promotes apoptosis in the A2058 human melanoma cell line expressing the oncogenic BRAF V600E mutation. Zeaxanthin 40 µM (IC50) induced chromatin condensation, nuclear blebbing, hypodiploidy, accumulation of cells in sub-G1 phase, DNA internucleosomal fragmentation and activation of caspase-3. Western blot analysis revealed that zeaxanthin induced up-regulation of the pro-apoptotic factors Bim and Bid and inhibition of NF-κB transactivation. Additionally, zeaxanthin sensitized A2058 melanoma cells in vitro to the cytotoxic activity of vemurafenib, a BRAF inhibitor widely used for the clinical management of melanoma, suggesting its potential interest as dietary adjuvant increasing melanoma cells sensitivity to chemotherapy.