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Apoptosis is regulated by interactions between the BH3-only and multi-domain Bcl-2 family proteins. These interactions are integrated on the outer mitochondrial membrane (OMM) where they set the threshold for apoptosis, known as mitochondrial priming. However, how mitochondrial priming is controlled at the level of single cells remains unclear. Retrotranslocation of Bcl-XL has been proposed as one mechanism, removing pro-apoptotic Bcl-2 proteins from the OMM, thus reducing priming. Contrary to this view, we now show that Bcl-XL retrotranslocation is inhibited by binding to its BH3-only partners, resulting in accumulation of these protein complexes on mitochondria. We find that Bcl-XL retrotranslocation dynamics are tightly coupled to mitochondrial priming. Quantifying these dynamics indicates the heterogeneity in priming between cells within a population and predicts how they subsequently respond to a pro-apoptotic signal.

Background Glioblastoma (GBM) has been extensively researched over the last few decades, yet despite aggressive multimodal treatment, recurrence is inevitable and second-line treatment options are limited. Here, we demonstrate how high-throughput screening (HTS) in multicellular spheroids can generate physiologically relevant patient chemosensitivity data using patient-derived cells in a rapid and cost-effective manner. Our HTS system identified actinomycin D (ACTD) to be highly cytotoxic over a panel of 12 patient-derived glioma stemlike cell (GSC) lines. ACTD is an antineoplastic antibiotic used in the treatment of childhood cancers. Here, we validate ACTD as a potential repurposed therapeutic for GBM in 3-dimensional GSC cultures and patient-derived xenograft models of recurrent glioblastoma. Methods Twelve patient-derived GSC lines were screened at 10 µM, as multicellular spheroids, in a 384-well serum-free assay with 133 FDA-approved compounds. GSCs were then treated in vitro with ACTD at established half-maximal inhibitory concentrations (IC50). Downregulation of sex determining region Y–box 2 (Sox2), a stem cell transcription factor, was investigated via western blot and through immunohistological assessment of murine brain tissue. Results Treatment with ACTD was shown to significantly reduce tumor growth in 2 recurrent GBM patient-derived models and significantly increased survival. ACTD is also shown to specifically downregulate the expression of Sox2 both in vitro and in vivo. Conclusion These findings indicate that, as predicted by our HTS, ACTD could deplete the cancer stem cell population within the tumor mass, ultimately leading to a delay in tumor progression. Key Points 1. High-throughput chemosensitivity data demonstrated the broad efficacy of actinomycin D, which was validated in 3 preclinical models of glioblastoma. 2. Actinomycin D downregulated Sox2 in vitro and in vivo, indicating that this agent could target the stem cell population of GBM tumors., info:eu-repo/semantics/published

Introduction: SMAD4, the central mediator of the Transforming Growth-Beta (TGF-Beta) signaling pathway, is inactivated in 50-55% of pancreatic ductal adenocarcinomas (PDACs). SMAD4 loss of expression has been described as a negative prognostic factor in PDAC and associated with increased rate of metastasis and resistance to adjuvant therapy. However, the impact of SMAD4 inactivation in patients receiving neoadjuvant therapy (NAT) is not well characterized. The aim of our study is to investigate if SMAD4 loss is a prognostic factor in a cohort of patients who underwent surgery for PDAC and, more specifically, in patients receiving NAT. Material and methods: We analyzed a single center retrospective series of 204 patients who underwent surgical resection for PDAC between 2004-2021. SMAD4 nuclear expression was semi-quantitively assessed by immunohistochemistry (IHC) on tumor samples and related to clinical variables including overall survival (OS) and disease free survival (DFS). Results: Among the 204 patients, 64 (31,37%) received NAT and 140 (68,63%) did not. In the NAT cohort, 33 (51,47%) tumors were SMAD4-positive and 31 (48,43%) were SMAD4-negative and in the non-NAT group, 84 (60%) and 56 (40%) patients presented SMAD4-positive and -negative tumors, respectively (p=0.28). Both in the NAT and non-NAT cohort of patients, SMAD4 status was not associated with clinico-pathological variables and did not impact OS. However, regarding DFS, in patients without NAT, SMAD4 loss seems to be associated with shorter DFS (p=0.073). In a multivariate analysis including the significant clinico-pathological variables, SMAD4 status showed a tendency to remain an independent prognostic variable (p=0.052). Moreover, in patients that received NAT, univariate analysis indicated that SMAD4 loss was associated with worse DFS (p=0.018). However, in a multivariate analysis, only vascular invasion (p=0.02) and the subtype of administrated NAT (FOLFIRINOX or GEMCITABINE-based) (p=0.0004) were independent prognostic factors for DFS. We therefore evaluated the prognostic significance of SMAD4 loss in a homogenous group of patients receiving FOLFIRINOX-based therapy (n=45). Univariate and multivariate analysis indicated that in these patients, SMAD4 loss was an independent negative prognostic factor (p=0.009) as well as vascular invasion (p=0.003). Conclusion: This study highlights the potential prognostic role of SMAD4 loss in the DFS of PDAC patients, more specifically in patients receiving NAT. Citation Format: Marie-Lucie Racu, Dana Bernardi, Aniss Chaouche, Egor Zindy, Julie Navez, Patrizia Loi, Calliope Maris, Jean Closset, Jean-Luc Van Laethem, Christine Decaestecker, Isabelle Salmon, Nicky D'Haene. Impact of SMAD4 loss in patients with pancreatic ductal adenocarcinoma receiving neoadjuvant therapy [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B043.

Saliva secretion requires effective translocation of aquaporin 5 (AQP5) water channel to the salivary glands (SGs) acinar apical membrane. Patients with Sjögren’s syndrome (SS) display abnormal AQP5 localization within acinar cells from SGs that correlate with sicca manifestation and glands hypofunction. Several proteins such as Prolactin-inducible protein (PIP) may regulate AQP5 trafficking as observed in lacrimal glands from mice. However, the role of the AQP5-PIP complex remains poorly understood. In the present study, we show that PIP interacts with AQP5 in vitro and in mice as well as in human SGs and that PIP misexpression correlates with an altered AQP5 distribution at the acinar apical membrane in PIP knockout mice and SS hMSG. Furthermore, our data show that the protein-protein interaction involves the AQP5 C-terminus and the N-terminal of PIP (one molecule of PIP per AQP5 tetramer). In conclusion, our findings highlight for the first time the role of PIP as a protein controlling AQP5 localization in human salivary glands but extend beyond due to the PIP-AQP5 interaction described in lung and breast cancers.

Sjögren’s syndrome (SS) is an exocrinopathy characterized by the hypofunction of salivary glands (SGs). Aquaporin-5 (AQP5), a water channel involved in saliva formation, is aberrantly distributed in SS SG acini and contributes to glandular dysfunction. We aimed to investigate the role of ezrin in AQP5 mislocalization in SS SGs. The AQP5–ezrin interaction was assessed by immunoprecipitation and proteome analysis and by proximity ligation assay in immortalized human SG cells. We demonstrated, for the first time, an interaction between ezrin and AQP5. A model of the complex was derived by computer modeling and in silico docking, suggesting that AQP5 interacts with the ezrin FERM-domain via its C-terminus. The interaction was also investigated in human minor salivary gland (hMSG) acini from SS patients (SICCA-SS), showing that AQP5–ezrin complexes were absent or mislocalized to the basolateral side of SG acini rather than the apical region compared to controls (SICCA-NS). Furthermore, in SICCA-SS hMSG acinar cells, ezrin immunoreactivity was decreased at the acinar apical region and higher at basal or lateral regions, accounting for altered AQP5–ezrin co-localization. Our data reveal that AQP5–ezrin interactions in human SGs could be involved in the regulation of AQP5 trafficking and may contribute to AQP5-altered localization in SS patients

Integration of signalling downstream of individual receptor tyrosine kinases (RTKs) is crucial to fine-tune cellular homeostasis during development and in pathological conditions, including breast cancer. However, how signalling integration is regulated and whether the endocytic fate of single receptors controls such signalling integration remains poorly elucidated. Combining quantitative phosphoproteomics and targeted assays, we generated a detailed picture of recycling-dependent fibroblast growth factor (FGF) signalling in breast cancer cells, with a focus on distinct FGF receptors (FGFRs). We discovered reciprocal priming between FGFRs and epidermal growth factor (EGF) receptor (EGFR) that is coordinated at recycling endosomes. FGFR recycling ligands induce EGFR phosphorylation on threonine 693. This phosphorylation event alters both FGFR and EGFR trafficking and primes FGFR-mediated proliferation but not cell invasion. In turn, FGFR signalling primes EGF-mediated outputs via EGFR threonine 693 phosphorylation. This reciprocal priming between distinct families of RTKs from recycling endosomes exemplifies a novel signalling integration hub where recycling endosomes orchestrate cellular behaviour. Therefore, targeting reciprocal priming over individual receptors may improve personalized therapies in breast and other cancers.

SUMMARYIntegration of signalling downstream of individual receptor tyrosine kinases (RTKs) is crucial to fine tune cellular homeostasis during development and in pathological conditions, including breast cancer. However, how signalling integration is regulated and whether the endocytic fate of single receptors controls such signalling integration remains poorly elucidated. Combining quantitative phosphoproteomics and targeted assays, we generated a detailed picture of recycling-dependent fibroblast growth factor (FGF) signalling in breast cancer cells, with a focus on distinct FGF receptors (FGFRs). We discovered reciprocal priming between FGFRs and epidermal growth factor (EGF) receptor (EGFR) that is coordinated at recycling endosomes. FGFR recycling ligands induce EGFR phosphorylation on threonine 693. This phosphorylation event alters both FGFR and EGFR trafficking and primes FGFR-mediated proliferation but not cell invasion. In turn, FGFR signalling primes EGF-mediated outputs via EGFR threonine 693 phosphorylation. This reciprocal priming between distinct families of RTKs from recycling endosomes exemplifies a novel signalling integration hub where recycling endosomes orchestrate cellular behaviour. Therefore, targeting reciprocal priming over individual receptors may improve personalized therapies in breast and other cancers.

SummaryMicroenvironmental stiffness regulates the behaviour of both normal and cancer cells. In breast tissue, high mammographic density (HMD), which reflects greater organisation and stiffness of the periductal collagen, represents a significant risk factor for cancer. However, the mechanistic link between extracellular matrix (ECM) stiffness and increased risk of breast tumour initiation remains unclear. In particular, how increased ECM stiffness might promote genomic damage, leading to the acquisition of transforming mutations, remains to be determined. Here we determine that ECM stiffness induces changes in mammary epithelial cell (MEC) metabolism that drive genomic damage. Using a 3D-culture model, we demonstrate that genome-wide transcriptional changes in response to increased ECM stiffness impair the ability of MECs to remove reactive aldehyde species, resulting in greater accumulation of DNA damage in a RhoA-dependent manner. Together, our results provide a mechanistic link between increased ECM stiffness and the genomic damage required for breast cancer initiation.

Glucocorticoids (GCs) act through the glucocorticoid receptor (GR, also known as NR3C1) to regulate immunity, energy metabolism and tissue repair. Upon ligand binding, activated GR mediates cellular effects by regulating gene expression, but some GR effects can occur rapidly without new transcription. Here, we show that GCs rapidly inhibit cell migration, in response to both GR agonist and antagonist ligand binding. The inhibitory effect on migration is prevented by GR knockdown with siRNA, confirming GR specificity, but not by actinomycin D treatment, suggesting a non-transcriptional mechanism. We identified a rapid onset increase in microtubule polymerisation following GC treatment, identifying cytoskeletal stabilisation as the likely mechanism of action. HDAC6 overexpression, but not knockdown of αTAT1, rescued the GC effect, implicating HDAC6 as the GR effector. Consistent with this hypothesis, ligand-dependent cytoplasmic interaction between GR and HDAC6 was demonstrated by quantitative imaging. Taken together, we propose that activated GR inhibits HDAC6 function, and thereby increases the stability of the microtubule network to reduce cell motility. We therefore report a novel, non-transcriptional mechanism whereby GCs impair cell motility through inhibition of HDAC6 and rapid reorganization of the cell architecture. This article has an associated First Person interview with the first author of the paper., Summary: A novel inhibitory mechanism of glucocorticoid (GC) action on cell migration is dependent on the α-tubulin deacetylase HDAC6, which has implications for the design of novel GCs with improved therapeutic benefit.

Evolutionarily conserved circadian clocks generate 24-hour rhythms in physiology and behaviour that adapt organisms to their daily and seasonal environments. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus is the principal co-ordinator of the cell-autonomous clocks distributed across all major tissues. The importance of robust daily rhythms is highlighted by experimental and epidemiological associations between circadian disruption and human diseases. BMAL1 (a bHLH-PAS domain-containing transcription factor) is the master positive regulator within the transcriptional-translational feedback loops (TTFLs) that cell-autonomously define circadian time. It drives transcription of the negative regulators Period and Cryptochrome alongside numerous clock output genes, and thereby powers circadian time-keeping. Because deletion of Bmal1 alone is sufficient to eliminate circadian rhythms in cells and the whole animal it has been widely used as a model for molecular disruption of circadian rhythms, revealing essential, tissue-specific roles of BMAL1 in, for example, the brain, liver and the musculoskeletal system. Moreover, BMAL1 has clock-independent functions that influence ageing and protein translation. Despite the essential role of BMAL1 in circadian time-keeping, direct measures of its intra-cellular behaviour are still lacking. To fill this knowledge-gap, we used CRISPR Cas9 to generate a mouse expressing a knock-in fluorescent fusion of endogenous BMAL1 protein (Venus::BMAL1) for quantitative live imaging in physiological settings. The Bmal1Venus mouse model enabled us to visualise and quantify the daily behaviour of this core clock factor in central (SCN) and peripheral clocks, with single-cell resolution that revealed its circadian expression, anti-phasic to negative regulators, nuclear-cytoplasmic mobility and molecular abundance., Author summary Cell-autonomous circadian clocks are transcriptional/translational feedback loops that co-ordinate almost all mammalian physiology and behaviour. Although their genetic basis is well understood, we are largely ignorant of the natural behaviour of clock proteins and how they work within these loops. This is particularly true for the essential transcriptional activator BMAL1. To address this, we created and validated a mouse carrying a fully functional knock-in allele that encodes a fluorescent fusion of BMAL1 (Venus::BMAL1). Quantitative live imaging in tissue explants and cells, including the central clock of the suprachiasmatic nucleus (SCN), revealed the circadian expression, nuclear-cytoplasmic mobility, fast kinetics and surprisingly low molecular abundance of endogenous BMAL1, providing significant quantitative insights into the intracellular mechanisms of circadian timing at single-cell resolution.

We have shown using SEEC microscopy that avidin addition to Biotinyl-Cap-PE lipids forms clusters, reducing the levels of association. By using PDP-PE lipids with PEG linkers, we have achieved much higher avidin levels with no clustering effects., info:eu-repo/semantics/published

Small proteinaceous oligomeric species contribute to the formation of larger aggregates, a phenomenon that is of direct relevance to the characterization of protein aggregation in biopharmaceuticals and understanding the underlying processes contributing to neurodegenerative diseases.The ability to monitor in situ oligomerization and aggregation processes renders imaging and image analysis an attractive approach for gaining a mechanistic insight into early processes contributing to the formation of larger aggregates in disease models and biologics. The combination of image analysis tools enables the detection of both oligomeric and larger aggregate subtype in contrast to conventional kinetic-based approaches that lack the ability to resolve dimers from monomeric moieties in samples containing mixed populations.In this chapter, we describe the process for confocal time series image acquisition for monitoring the in situ loss of monomers, and the subsequent analysis pipeline using spatial intensity distribution analysis (SpIDA) to evaluate oligomer content.

Small proteinaceous oligomeric species contribute to the formation of larger aggregates, a phenomenon that is of direct relevance to the characterization of protein aggregation in biopharmaceuticals and understanding the underlying processes contributing to neurodegenerative diseases. The ability to monitor in situ oligomerization and aggregation processes renders imaging and image analysis an attractive approach for gaining a mechanistic insight into early processes contributing to the formation of larger aggregates in disease models and biologics. The combination of image analysis tools enables the detection of both oligomeric and larger aggregate subtype in contrast to conventional kinetic-based approaches that lack the ability to resolve dimers from monomeric moieties in samples containing mixed populations. In this chapter, we describe the process for confocal time series image acquisition for monitoring the in situ loss of monomers, and the subsequent analysis pipeline using spatial intensity distribution analysis (SpIDA) to evaluate oligomer content.

Background Circadian rhythms maintain tissue homeostasis during the 24-h day-night cycle. Cell-autonomous circadian clocks play fundamental roles in cell division, DNA damage responses and metabolism. Circadian disruptions have been proposed as a contributing factor for cancer initiation and progression, although definitive evidence for altered molecular circadian clocks in cancer is still lacking. In this study, we looked at circadian clocks in breast cancer. Methods We isolated primary tumours and normal tissues from the same individuals who had developed breast cancer with no metastases. We assessed circadian clocks within primary cells of the patients by lentiviral expression of circadian reporters, and the levels of clock genes in tissues by qPCR. We histologically examined collagen organisation within the normal and tumour tissue areas, and probed the stiffness of the stroma adjacent to normal and tumour epithelium using atomic force microscopy. Results Epithelial ducts were disorganised within the tumour areas. Circadian clocks were altered in cultured tumour cells. Tumour regions were surrounded by stroma with an altered collagen organisation and increased stiffness. Levels of Bmal1 messenger RNA (mRNA) were significantly altered in the tumours in comparison to normal epithelia. Conclusion Circadian rhythms are suppressed in breast tumour epithelia in comparison to the normal epithelia in paired patient samples. This correlates with increased tissue stiffness around the tumour region. We suggest possible involvement of altered circadian clocks in the development and progression of breast cancer. Electronic supplementary material The online version of this article (10.1186/s13058-018-1053-4) contains supplementary material, which is available to authorized users.

The formation of uniaxial fibrous tissues with defined viscoelastic properties implies the existence of an orchestrated mechanical interaction between the cytoskeleton and the extracellular matrix. This study addresses the nature of this interaction. The hypothesis is that this mechanical interplay underpins the mechanical development of the tissue. In embryonic tendon tissue, an early event in the development of a mechanically robust tissue is the interaction of the pointed tips of extracellular collagen fibrils with the fibroblast plasma membrane to form stable interface structures (fibripositors). Here, we used a fibroblast-generated tissue that is structurally and mechanically matched to embryonic tendon to demonstrate homeostasis of cell-derived and external strain-derived tension over repeated cycles of strain and relaxation. A cell-derived oscillatory tension component is evident in this matrix construct. This oscillatory tension involves synchronization of individual cell forces across the construct and is induced in each strain cycle by transient relaxation and transient tensioning of the tissue. The cell-derived tension along with the oscillatory component is absent in the presence of blebbistatin, which disrupts actinomyosin force generation of the cell. The time period of this oscillation (60-90 s) is well-defined in each tissue sample and matches a primary viscoelastic relaxation time. We hypothesize that this mechanical oscillation of fibroblasts with plasma membrane anchored collagen fibrils is a key factor in mechanical sensing and feedback regulation in the formation of tensile tissues.

Rab-coupling protein–mediated integrin trafficking promotes filopodia formation via RhoA-ROCK-FHOD3, generating non-lamellipodial actin spike protrusions that drive cancer cell migration in 3D extracellular matrix and in vivo., Invasive migration in 3D extracellular matrix (ECM) is crucial to cancer metastasis, yet little is known of the molecular mechanisms that drive reorganization of the cytoskeleton as cancer cells disseminate in vivo. 2D Rac-driven lamellipodial migration is well understood, but how these features apply to 3D migration is not clear. We find that lamellipodia-like protrusions and retrograde actin flow are indeed observed in cells moving in 3D ECM. However, Rab-coupling protein (RCP)-driven endocytic recycling of α5β1 integrin enhances invasive migration of cancer cells into fibronectin-rich 3D ECM, driven by RhoA and filopodial spike-based protrusions, not lamellipodia. Furthermore, we show that actin spike protrusions are Arp2/3-independent. Dynamic actin spike assembly in cells invading in vitro and in vivo is regulated by Formin homology-2 domain containing 3 (FHOD3), which is activated by RhoA/ROCK, establishing a novel mechanism through which the RCP–α5β1 pathway reprograms the actin cytoskeleton to promote invasive migration and local invasion in vivo.

The circadian clock is an autonomous molecular feedback loop inside almost every cell in the body. We have shown that the mammary epithelial circadian clock is regulated by the cellular microenvironment. Moreover, a stiff extracellular matrix dampens the oscillations of the epithelial molecular clock. Here, we extend this analysis to other tissues and cell types, and identify an inverse relationship between circadian clocks in epithelia and fibroblasts. Epithelial cells from mammary gland, lung and skin have significantly stronger oscillations of clock genes in soft 3D microenvironments, compared to stiff 2D environments. Fibroblasts isolated from the same tissues show the opposite response, exhibiting stronger oscillations and more prolonged rhythmicity in stiff microenvironments. RNA analysis identified that a subset of mammary epithelial clock genes, and their regulators, are upregulated in 3D microenvironments in soft compared to stiff gels. Furthermore, the same genes are inversely regulated in fibroblasts isolated from the same tissues. Thus, our data reveal for the first time an intrinsic difference in the regulation of circadian genes in epithelia and fibroblasts., Highlighted Article: Cell-matrix interactions are known to control circadian clocks. Here, we discovered that the matrix inversely regulates epithelia and fibroblasts.

Cell migration in 3D microenvironments is fundamental to development, homeostasis and the pathobiology of diseases such as cancer. Rab-coupling protein (RCP) dependent co-trafficking of α5β1 and EGFR1 promotes cancer cell invasion into fibronectin (FN) containing extracellular matrix (ECM), by potentiating EGFR1 signalling at the front of invasive cells. This promotes a switch in RhoGTPase signalling to inhibit Rac1 and activate a RhoA-ROCK-Formin homology domain-containing 3 (FHOD3) pathway and generate filopodial actin-spike protrusions which drive invasion. To further understand the signalling network that drives RCP-driven invasive migration, we generated a Boolean logical model based on existing network pathways/models, where each node can be interrogated by computational simulation. The model predicted an unanticipated feedback loop, whereby Raf/MEK/ERK signalling maintains suppression of Rac1 by inhibiting the Rac-activating Sos1-Eps8-Abi1 complex, allowing RhoA activity to predominate in invasive protrusions. MEK inhibition was sufficient to promote lamellipodia formation and oppose filopodial actin-spike formation, and led to activation of Rac and inactivation of RhoA at the leading edge of cells moving in 3D matrix. Furthermore, MEK inhibition abrogated RCP/α5β1/EGFR1-driven invasive migration. However, upon knockdown of Eps8 (to suppress the Sos1-Abi1-Eps8 complex), MEK inhibition had no effect on RhoGTPase activity and did not oppose invasive migration, suggesting that MEK-ERK signalling suppresses the Rac-activating Sos1-Abi1-Eps8 complex to maintain RhoA activity and promote filopodial actin-spike formation and invasive migration. Our study highlights the predictive potential of mathematical modelling approaches, and demonstrates that a simple intervention (MEK-inhibition) could be of therapeutic benefit in preventing invasive migration and metastasis., Author Summary The majority of cancer-related fatalities are caused by the movement of cancer cells away from the primary site to form metastases, making understanding the signalling mechanisms which underpin cell migration and invasion through their local environment of paramount importance. Much has been discovered about key events leading to invasive cell migration. Here, we have taken this prior knowledge to build a powerful predictive model based on simple ON/OFF relations and logic to determine potential intervention targets to reduce harmful invasive migration. Interrogating our model, we have identified a negative feedback loop important to the signalling that determines invasive migration, the breaking of which reverts cells to a slower, less invasive phenotype. We have supported this feedback loop prediction using an array of in vitro experiments performed in cells within 2-D and physiologically relevant 3-D environments. Our findings demonstrate the predictive power of such modelling techniques, and could form the basis for clinical intervention to prevent metastasis in certain cancers.

The mechanical properties of the cell nucleus change to allow cells to migrate, but how chromatin modifications contribute to nuclear deformability has not been defined. Here, we demonstrate that a major factor in this process involves epigenetic changes that underpin nuclear structure. We investigated the link between cell adhesion and epigenetic changes in T-cells, and demonstrate that T-cell adhesion to VCAM1 via α4β1 integrin drives histone H3 methylation (H3K9me2/3) through the methyltransferase G9a. In this process, active G9a is recruited to the nuclear envelope and interacts with lamin B1 during T-cell adhesion through α4β1 integrin. G9a activity not only reorganises the chromatin structure in T-cells, but also affects the stiffness and viscoelastic properties of the nucleus. Moreover, we further demonstrated that these epigenetic changes were linked to lymphocyte movement, as depletion or inhibition of G9a blocks T-cell migration in both 2D and 3D environments. Thus, our results identify a novel mechanism in T-cells by which α4β1 integrin signaling drives specific chromatin modifications, which alter the physical properties of the nucleus and thereby enable T-cell migration.

Real-time confocal imaging was utilised to monitor the in situ loss of BSA monomers and aggregate formation using Spatial Intensity Distribution Analysis (SpIDA) and Raster Image Correlation Spectroscopy (RICS). At the proof of concept level this work has demonstrated the applicability of RICS and SpIDA for monitoring protein oligomerisation and larger aggregate formation.

Recently, the multifunctional peptide TatLK15 resulting from the fusion of the cell penetrating peptide Tat and the amphipathic peptide LK15 was shown to be efficient at mediating siRNA and shRNA delivery in leukemia cells to silence the bcr-abl oncoprotein. The present study focused on TatLK15 peptide cellular uptake and defining conditions for its use within a range of doses exhibiting minimal toxicity. The initial part of the study carried out in solution confirmed that the insertion of a glycine bridge allowed retention of the LK15 α-helicity, and fluorescence correlation spectroscopy did not reveal preferential conformations at the studied concentrations. In the second part, TatLK15 uptake mechanisms appeared peptide dose- and cell line- dependent as well as requiring membrane potential. Below a critical dose, TatLK15 toxicity appeared limited for approximately three hours as demonstrated by the combined use of lactate dehydrogenase release, MTT assays, and time-dependent observation of membrane-impermeant dye uptake using high content screening apparatus. Furthermore, toxicity was observed to occur rapidly at higher peptide doses. Finally, a comparison between TatLK15 and another Tat amphipathic peptide construct suggested that α-helix content should be viewed as a key element in the development of similar peptides. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:293–304, 2014

The application of raster image correlation spectroscopy (RICS) as a tool for the characterisation of protein diffusion was assessed using a model protein, bovine serum albumin (BSA), as a function of formulation and denaturing conditions. RICS results were also validated against dynamic light scattering and fluorescence correlation spectroscopy. Results from this study demonstrate correlation between outputs obtained from the three experimental techniques. Ionic strength independency was observed at pH 7, and a reduction in the corresponding diffusion coefficients was noted at pH 4.5 for 1 µM BSA-Alexa Fluor 488. Conversely, at pH 5.2, higher-concentration samples exhibited ionic strength dependency. Buffer composition, sample pretreatment, thermal denaturation and freeze-thaw cycling were also found to influence RICS output, with a reduction in the diffusion coefficient and the number of particles observed for both pH values. In conclusion, RICS analysis of images acquired using a commercial confocal microscope offers a potential scope for application to both quantitative and qualitative characterisation of macromolecular behaviour in solution.