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Ovarian cancer is one of the most lethal gynaecological cancer. Heterogeneity associated with this cancer can be partially attributed to cancer cells being highly multinucleated and polyploid in nature. Stress-induced cytotoxic effects of cisplatin - a chemotherapy drug used to treat ovarian cancer, on subcellular organelles including the ER, mitochondria, nucleus and the cytoskeleton are well known, however, its effect on the structural integrity of the Golgi apparatus and associated alterations in functions are not reported. In this study, the effect of cisplatin on the structure and function of the Golgi apparatus are examined and a novel pathway for cisplatin-induced changes in the Golgi is also reported. Additionally, a mechanism is also proposed for formation of multinucleated ovarian cancer cells and a role for transcription adapter-3 (TADA3)-mediated multinucleation has been suggested. Treatment of ovarian cancer cells with cisplatin resulted in significant fragmentation of the Golgi apparatus with cisplatin-induced Golgi fragmentation detected before cisplatin-induced DNA damage. Inhibition of cisplatin-stress induced activation of c-Jun-N-terminal kinase (JNK) significantly reduced fragmentation of the Golgi in ovarian cancer cells while extracellular signal-regulated kinase (ERK1/2) inhibition significantly increased fragmentation in A2780 cells but had no effect on other cell lines (OVCAR3 and SKOV3) tested. Cells exposed to cisplatin during migration showed significantly depolarised Golgi, reduced cell motility and wound healing capacity. Protein trafficking was also significantly reduced in response to cisplatin. Cell-line specific effect was found on the expression of terminal GalNAc glycans in response to cisplatin. Moreover, ovarian cancer cells were observed to fuse with each other following cell division producing multinucleated cells. Gene expression correlation analysis identified TADA3 as a candidate gene associated with multinuclearity. TADA3

Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport and cellular signalling. For these proteins to function effectively there needs to be structure within the PM allowing, for example, proteins in the same signalling cascade to be spatially organized. Here we demonstrate that several proteins with divergent functions are located in clusters of differing size in the membrane using sub-diffraction-limited Airyscan confocal microscopy. Single particle tracking reveals that these proteins move at different rates within the membrane. Actin and microtubule cytoskeletons appear to significantly regulate the mobility of one of these proteins (the pathogen receptor FLS2) and we further demonstrate that the cell wall is critical for the regulation of cluster size by quantifying single particle dynamics of proteins with key roles in morphogenesis (PIN3) and pathogen perception (FLS2). We propose a model in which the cell wall and cytoskeleton are pivotal for regulation of protein cluster size and dynamics thereby contributing to the formation and functionality of membrane nanodomains.

Extracellular vesicles (EVs) are small lipid bound spherical structures released by almost all types of cells. They are mediators of intercellular communication, however, the exact mechanisms of EV-cell interactions and uptake have not been fully characterised. EVs play key roles in cancer including all the stages of metastasis. Breast cancer is amongst the most common types of cancer for women and the primary cause of cancer related death. The high mortality rates are due to the metastatic spread of cancer cells and tumour recurrence after therapy. Studies have proposed chemotherapy to be a double-edged sword, which in some cases can promote metastasis. Here, it was hypothesised that EVs from chemotherapy treated cells were able to induce metastasis-related capacities to naïve cells. The aims of this project were: a) to explore the dynamics and specificity of EV uptake by recipient cells, and b) to investigate the effect of chemotherapy induced intercellular communication via EVs on breast cancer metastasis. To study EV uptake, different methods of EV labelling were tested. It was observed that PKH26 labelling may result in false-positive signals due to micelle formation. EV labelling with CFSE also produced non-specific effects, while dual labelling with both PKH26 and CFSE resulted in EV populations with heterogeneous labelling. EVs bearing a fluorescently tagged EV marker (mEmerald-CD81) were preferential for experiments investigating EV dynamics, as labelling appeared to be more specific than the dyes previously tested. Using this type of labelled EVs, it was shown that EV uptake differs across cell lines and the EV origin affects docking on the plasma membrane. Using a range of uptake inhibitors, it was observed that cholesterol may be involved in early EV-plasma membrane interactions, thus affecting EV uptake. Single molecule tracking revealed two patterns of such interactions: short and fast, and long and slow. To study the effect of EVs after chemotherapy

In plant cells, molecular connections link the cell wall–plasma membrane–actin cytoskeleton to form a continuum. It is hypothesized that the cell wall provides stable anchor points around which the actin cytoskeleton remodels. Here we use live cell imaging of fluorescently labelled marker proteins to quantify the organization and dynamics of the actin cytoskeleton and to determine the impact of disrupting connections within the continuum. Labelling of the actin cytoskeleton with green fluorescent protein (GFP)–fimbrin actin-binding domain 2 (FABD2) resulted in a network composed of fine filaments and thicker bundles that appeared as a highly dynamic remodelling meshwork. This differed substantially from the GFP–Lifeact-labelled network that appeared much more sparse with thick bundles that underwent ‘simple movement’, in which the bundles slightly change position, but in such a manner that the structure of the network was not substantially altered during the time of observation. Label-dependent differences in actin network morphology and remodelling necessitated development of two new image analysis techniques. The first of these, ‘pairwise image subtraction’, was applied to measurement of the more rapidly remodelling actin network labelled with GFP–FABD2, while the second, ‘cumulative fluorescence intensity’, was used to measure bulk remodelling of the actin cytoskeleton when labelled with GFP–Lifeact. In each case, these analysis techniques show that the actin cytoskeleton has a decreased rate of bulk remodelling when the cell wall–plasma membrane–actin continuum is disrupted either by plasmolysis or with isoxaben, a drug that specifically inhibits cellulose deposition. Changes in the rate of actin remodelling also affect its functionality, as observed by alteration in Golgi body motility.

Rhizosecretion of recombinant pharmaceuticals from in vitro hydroponic transgenic plant cultures is a simple, low cost, reproducible and controllable production method. Here, we demonstrate the application and adaptation of this manufacturing platform to a human antivitronectin IgG1 monoclonal antibody (mAb) called M12. The rationale for specific growth medium additives was established by phenotypic analysis of root structure and by LC-ESI-MS/MS profiling of the total protein content profile of the hydroponic medium. Through a combination of optimization approaches, mAb yields in hydroponic medium reached 46 μg/mL in 1 week, the highest figure reported for a recombinant mAb in a plant secretion-based system to date. The rhizosecretome was determined to contain 104 proteins, with the mAb heavy and light chains the most abundant. This enabled evaluation of a simple, scalable extraction and purification protocol and demonstration that only minimal processing was necessary prior to protein A affinity chromatography. MALDI-TOF MS revealed that purified mAb contained predominantly complex-type plant N-glycans, in three major glycoforms. The binding of M12 purified from hydroponic medium to vitronectin was comparable to its Chinese hamster ovary (CHO)-derived counterpart. This study demonstrates that in vitro hydroponic cultivation coupled with recombinant protein rhizosecretion can be a practical, low-cost production platform for monoclonal antibodies.

This study used live cell imaging of fluorescently labelled marker proteins to quantify the organisation and dynamics of the actin cytoskeleton and to determine if and how it was affected when the connection between the cell wall and plasma membrane was disturbed. Two image analysis techniques were developed to measure the dynamic remodelling of the actin cytoskeleton: Pairwise image subtraction and Cumulative fluorescence intensity. Both these analysis techniques showed that the actin cytoskeleton had a decreased rate of bulk remodelling when the cell wall-plasma membrane-actin continuum was disrupted either by plasmolysis or with isoxaben, a drug that specifically inhibits cellulose deposition. Changes in the rate of actin remodelling also affected its functionality as observed by alteration in Golgi body motility. The possibility that the changes seen were part of a general abiotic stress response by the actin cytoskeleton was refuted by showing that temperature stress did not decrease the rate of bulk remodelling. The effect of complete cell wall removal on the actin cytoskeleton was also investigated, but there was little difference in bulk remodelling between protoplasts immediately post-cell wall removal and after 24 h of cell wall re-growth. In addition to actin cytoskeleton remodelling, this study also aimed to discover if plasma membrane protein mobility was altered in cells with a perturbed cell wall. It is known that the mobility of plasma membrane proteins can be increased in plant cells that have had their cell wall removed or separated from the plasma membrane. In this study, cell wall perturbations were caused by mutations leading to a lack of detectable xyloglucan. Some of the plasma membrane proteins examined had altered mobility in cells with a disturbed cell wall, but others did not, suggesting different components in the cell wall affect specific plasma membrane associations. Together these results show that disturbing the cell wall-plasma membr