Optimising a self-assembling peptide hydrogel as a Matrigel alternative for 3-dimensional mammary epithelial cell culture.

Three-dimensional (3D) organoid models have been instrumental in understanding molecular mechanisms responsible for many cellular processes and diseases. However, established organic biomaterial scaffolds used for 3D hydrogel cultures, such as Matrigel, are biochemically complex and display significant batch variability, limiting reproducibility in experiments. Recently, there has been significant progress in the development of synthetic hydrogels for in vitro cell culture that are reproducible, mechanically tuneable, and biocompatible.

Apoptotic priming is defined by the dynamic exchange of Bcl-2 proteins between mitochondria and cytosol.

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.

Mitochondrial dynamics regulate genome stability via control of caspase-dependent DNA damage.

Mitochondrial dysfunction is interconnected with cancer. Nevertheless, how defective mitochondria promote cancer is poorly understood. We find that mitochondrial dysfunction promotes DNA damage under conditions of increased apoptotic priming.

Elevated EDAR signalling promotes mammary gland tumourigenesis with squamous metaplasia.

Ectodysplasin A receptor (EDAR) is a death receptor in the Tumour Necrosis Factor Receptor (TNFR) superfamily with roles in the development of hair follicles, teeth and cutaneous glands. Here we report that human Oestrogen Receptor (ER) negative breast carcinomas which display squamous differentiation express EDAR strongly. Using a mouse model with a high Edar copy number, we show that elevated EDAR signalling results in a high incidence of mammary tumours in breeding female mice.

BioID-based proteomic analysis of the Bid interactome identifies novel proteins involved in cell-cycle-dependent apoptotic priming.

Apoptotic priming controls the commitment of cells to apoptosis by determining how close they lie to mitochondrial permeabilisation. Variations in priming are important for how both healthy and cancer cells respond to chemotherapeutic agents, but how it is dynamically coordinated by Bcl-2 proteins remains unclear. The Bcl-2 family protein Bid is phosphorylated when cells enter mitosis, increasing apoptotic priming and sensitivity to antimitotic drugs.

Mitochondrial residence of the apoptosis inducer BAX is more important than BAX oligomerization in promoting membrane permeabilization.

Permeabilization of the mitochondrial outer membrane is a key step in the intrinsic apoptosis pathway, triggered by the release of mitochondrial intermembrane space proteins into the cytoplasm. The BCL-2-associated X apoptosis regulator (BAX) protein critically contributes to this process by forming pores in the mitochondrial outer membrane. However, the relative roles of the mitochondrial residence of BAX and its oligomerization in promoting membrane permeabilization are unclear.

Vinculins interaction with talin is essential for mammary epithelial differentiation.

Vinculin is an essential component of cell adhesion complexes, where it regulates the strength and stability of adhesions. Whilst the role of vinculin in cell motility is well established, it remains unclear how vinculin contributes to other aspects of tissue function. Here we examine the role of vinculin in mammary epithelial cell phenotype.

Nuclear decoupling is part of a rapid protein-level cellular response to high-intensity mechanical loading.

Studies of cellular mechano-signaling have often utilized static models that do not fully replicate the dynamics of living tissues. Here, we examine the time-dependent response of primary human mesenchymal stem cells (hMSCs) to cyclic tensile strain (CTS). At low-intensity strain (1 h, 4% CTS at 1 Hz), cell characteristics mimic responses to increased substrate stiffness.

E2F1 interacts with BCL-xL and regulates its subcellular localization dynamics to trigger cell death.

E2F1 is the main pro-apoptotic effector of the pRB-regulated tumor suppressor pathway by promoting the transcription of various pro-apoptotic proteins. We report here that E2F1 partly localizes to mitochondria, where it favors mitochondrial outer membrane permeabilization. E2F1 interacts with BCL-xL independently from its BH3 binding interface and induces a stabilization of BCL-xL at mitochondrial membranes.

The requirement of integrins for breast epithelial proliferation.

Epithelial cells forming mammary gland ducts and alveoli require adhesion to the extracellular matrix for their function. Mammary epithelial cells need β1-integrins for normal cell cycle regulation. However, the role of β1-integrins in tumorigenesis has not been fully resolved.

Oncogenic activation of FAK drives apoptosis suppression in a 3D-culture model of breast cancer initiation.

A key hallmark of cancer cells is the loss of positional control over growth and survival. Focal adhesion kinase (FAK) is a tyrosine kinase localised at sites of integrin-mediated cell adhesion to the extracellular matrix. FAK controls a number of adhesion-dependent cellular functions, including migration, proliferation and survival.

The Integrin-Mediated ILK-Parvin-αPix Signaling Axis Controls Differentiation in Mammary Epithelial Cells.

Epithelial cell adhesion to the surrounding extracellular matrix is necessary for their proper behavior and function. During pregnancy and lactation, mammary epithelial cells (MECs) receive signals from their interaction with laminin via β1-integrin (β1-itg) to establish apico-basal polarity and to differentiate in response to prolactin. Downstream of β1-itg, the scaffold protein Integrin Linked Kinase (ILK) has been identified as the key signal transducer that is required for both lactational differentiation and the establishment of apico-basal polarity.

Mitosis and mitochondrial priming for apoptosis.

Cell division is a period of danger for cells, as inaccurate segregation of chromosomes can lead to loss of cell viability or aneuploidy. In order to protect against these dangers, cells ultimately initiate mitochondrial apoptosis if they are unable to correctly exit mitosis. A number of important chemotherapeutics exploit this response to delayed mitotic exit, but despite this, the molecular mechanism of the apoptotic timer in mitosis has proved elusive.

Axl tyrosine kinase protects against tubulo-interstitial apoptosis and progression of renal failure in a murine model of chronic kidney disease and hyperphosphataemia.

Chronic kidney disease (CKD) is defined as the progressive loss of renal function often involving glomerular, tubulo-interstitial and vascular pathology. CKD is associated with vascular calcification; the extent of which predicts morbidity and mortality. However, the molecular regulation of these events and the progression of chronic kidney disease are not fully elucidated.

Phosphorylation of the proapoptotic BH3-only protein bid primes mitochondria for apoptosis during mitotic arrest.

Mitosis is a moment of exquisite vulnerability for a metazoan cell. Failure to complete mitosis accurately can lead to aneuploidy and cancer initiation. Therefore, if the exit from mitosis is delayed, normal cells are usually removed by apoptosis.

Inhibitor of Apoptosis Proteins: Promising Targets for Cancer Therapy.

Cancer is a disease in which normal physiological processes are imbalanced, leading to tumour formation, metastasis and eventually death. Recent biological advances have led to the advent of targeted therapies to complement traditional chemotherapy and radiotherapy. However, a major problem still facing modern medicine is resistance to therapies, whether targeted or traditional.

Bax exists in a dynamic equilibrium between the cytosol and mitochondria to control apoptotic priming.

The proapoptotic Bcl-2 protein Bax is predominantly found in the cytosol of nonapoptotic cells and is commonly thought to translocate to mitochondria following an apoptotic stimulus. The current model for Bax activation is that BH3 proteins bind to cytosolic Bax, initiating mitochondrial targeting and outer-membrane permeabilization. Here, we challenge this and show that Bax is constitutively targeted to mitochondria but in nonapoptotic cells is constantly translocated back to the cytosol.

Bcl-2 proteins and mitochondria--specificity in membrane targeting for death.

The localization and control of Bcl-2 proteins on mitochondria is essential for the intrinsic pathway of apoptosis. Anti-apoptotic Bcl-2 proteins reside on the outer mitochondrial membrane (OMM) and prevent apoptosis by inhibiting the activation of the pro-apoptotic family members Bax and Bak. The Bcl-2 subfamily of BH3-only proteins can either inhibit the anti-apoptotic proteins or directly activate Bax or Bak.

Role for X-linked Inhibitor of apoptosis protein upstream of mitochondrial permeabilization.

Apoptosis is controlled by a signaling equilibrium between prosurvival and proapoptotic pathways, such that unwanted apoptosis is avoided, but when required it occurs rapidly and efficiently. Many apoptosis regulators display dual roles, depending upon whether a cell has received an apoptotic stimulus or not. Here, we identify a novel and unexpected function for X-linked inhibitor of apoptosis (XIAP) that occurs when apoptosis is triggered under physiological conditions.

How adhesion signals reach a mitochondrial conclusion--ECM regulation of apoptosis.

A fundamental aspect in metazoans is the ability of a cell to recognise its positional context within a tissue. This is important in both development and homeostasis, where cell proliferation, differentiation and apoptosis are strictly controlled to form and maintain tissues. Much information has been generated on how cells receive and interpret adhesion-mediated signals.

Notch activation induces Akt signaling via an autocrine loop to prevent apoptosis in breast epithelial cells.

The Notch pathway is aberrantly activated in a wide range of cancers, including breast carcinoma, and is required to maintain the transformed phenotype of many of these tumors. Notch signaling contributes to the transformed phenotype, in part, by preventing apoptosis in response to many different stimuli. However, it is unclear how Notch activation can lead to a general suppression of apoptosis.

Bid binding to negatively charged phospholipids may not be required for its pro-apoptotic activity in vivo.

Bid is a ubiquitous pro-apoptotic member of the Bcl-2 family that has been involved in a variety of pathways of cell death. Unique among pro-apoptotic proteins, Bid is activated after cleavage by the apical caspases of the extrinsic pathway; subsequently it moves to mitochondria, where it promotes the release of apoptogenic proteins in concert with other Bcl-2 family proteins like Bak. Diverse factors appear to modulate the pro-apoptotic action of Bid, from its avid binding to mitochondrial lipids (in particular, cardiolipin) to multiple phosphorylations at sites that can modulate its caspase cleavage.

FAK engages multiple pathways to maintain survival of fibroblasts and epithelia: differential roles for paxillin and p130Cas.

Different cell types interpret their distinct extracellular matrix (ECM) environments to bring about specific cell fate decisions, and can differentiate or undergo apoptosis depending on their local adhesive interactions. Apoptosis in response to an inappropriate ECM environment is termed ;anoikis', or homelessness. Several studies, utilising a variety of cell types, have indicated a common, crucial role for focal adhesion kinase (FAK) in suppressing anoikis.

Analysis of endogenous Bax complexes during apoptosis using blue native PAGE: implications for Bax activation and oligomerization.

Bax, a pro-apoptotic Bcl-2 family protein, translocates to mitochondria during apoptosis, where it causes MOMP (mitochondrial outer membrane permeabilization). MOMP releases pro-apoptotic factors, such as cytochrome c and SMAC (second mitochondrial activator of caspases)/Diablo, into the cytosol where they activate caspases. It is often inferred that Bax activation occurs in a single step, a conformational change in the protein causing its translocation and oligomerization into high-molecular-mass membrane pores.