Focus on Actin detection and Actin binding proteins

Actin can exist in two forms: Globular subunit (G-actin) and Filamentous polymer (F-actin). Both forms of actin interact with a plethora of proteins in the cell. To date there are over 50 distinct classes of Actin-Binding Proteins (ABPs), and the inventory is still far from complete. Actin Binding Proteins allow the actin cytoskeleton to respond rapidly to cellular and extracellular signals and are integral to cytoskeletal involvement in many cellular processes. These include cell shape and motility, muscle contraction, intracellular trafficking, cell pathogenesis and signal transduction.

In the coming weeks I’d like to give you an overview of methods in actin research with validated R&D products and kits (actin polymerisation, and G-F actin ratio detection in cells); I also invite you to take a look at a post recently released about actin visualization: Focus on Actin staining and visualization.

In today’s post, let’s concentrate on a method which allows measuring actin binding capabilities of proteins of interest. But it’s not only about the simple fact that a given protein is binding to actin, with the method presented here, you’re also able to get an idea of the functionality of the protein – be it F-actin bundling activity, F-actin severing activity or G-actin binding activity. [Read more…]

New way for derivation / maintenance of naïve human PSCs

Newly added to the Stemolecule portfolio are three small molecules which support a new approach for the derivation and maintenance of naïve human pluripotent stem cells. These three newly identified small molecules, WH-4-023, SB590885 and IM-12, are all kinase inhibitors.

small-molecules-03

ReproCELL is currently the only stem cell reagent company able to supply all components of the 5i/L/A (5 inhibitors/human LIF/Activin A) media supplement formulation needed for the derivation and maintenance of a “naïve” or ground-state of pluripotency of human cells as referenced in the October 2014 Cell Stem Cell paper by Theunissen et al. out of Dr. Rudolf Jaenisch’s Lab at MIT’s Whitehead Institute. The paper is titled “Systematic identification of culture conditions for induction and maintenance of naïve human pluripotency.”

Stemgent Stemolecule WH-4-023 (SRC Inhibitor)IM-12 Structure

Stemgent Stemolecule SB590885 (BRAF Inhibitor)

Stemgent Stemolecule IM-12 (GSK-3β Inhibitor)

 

The addition of these three new small molecules consolidates a 5i/L/A media supplement offering in one place. 5i/L/A media supplement consists of 5 kinase inhibitors; WH-4-023 SRC inhibitor, SB590885 BRAF inhibitor, IM-12 GSK-3β inhibitor, PD0325901 MEK inhibitor, Y27632 ROCK inhibitor, recombinant human leukemia inhibitory factor (LIF) and Activin A. The 5i/L/A media supplement allows for the derivation of and conversion of human PSCs to a naïve state of pluripotency hypothesized to be the human equivalent to the mouse ground state of pluripotency.

The Theunissen paper demonstrates the conversion of traditional human epiblastic pluripotent stem cells to a ground state of pluripotency thought to be equivalent to the traditional mouse embryonic stem cell state. The human equivalent to the mouse ground state of pluripotency is termed naïve. Naïve human PSCs differ from traditional human epiblastic ESCs in that human ESCs are FGF/BMP signaling dependent (equivalent to mouse epiblastic stem cells). While naïve PSCs are LIF/Stat3 signaling dependent (equivalent to traditional mouse ESCs). Both traditional mouse ESCs and naïve human PSCs demonstrate higher single cell viability when passaging. Likewise both cell types demonstrate enhanced proliferation when compared to epiblastic stem cells. These attributes make naïve human pluripotent stem cells a good choice for genetic manipulation and gene targeting applications.

Related products used in the paper that researchers may be interested in include: Human recombinant FGF-basic growth factor, CHIR99021 and doxycycline.

Focus on Actin staining and visualization

Actin serves as one of the major cytoskeleton structures. It is a crucial component involved in a plethora of processes in cell biology:  stabilizing the cell shape, cell movements (e.g. cell migration)  and intracellular movements and transport mechanisms.

Actin is a 43 kDa protein that is very highly conserved between species. Actin has three main isotypes (α-actin, β-actin and γ-actin), which show >90% amino-acid (aa) homology between isotypes and >98% homology within members of a particular isotypic group.

A brief reminder: G-actin polymerizes to form F-actin

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Fig. 1: Double-helical structure of actin filaments (provided by Cytoskeleton Inc.)

Globular-actin (G-actin) readily polymerizes under physiological conditions to form Filamentous-actin (F-actin) with the concomitant hydrolysis of ATP. F-actin is a double-helical filament (Fig. 1).  Actin can polymerize from both ends in vitro. However, the rate of polymerization is not equal. This results in an intrinsic polarity in the actin filament. It has therefore become the convention to term the rapidly polymerizing end the plus-end or barbed-end (+) while the slow growing end is called the minus-end or pointed-end (-).

In the coming weeks I will give you an overview about methods in actin research with validated R&D products and kits (actin binding and actin binding proteins, actin polymerisation, and G-F actin ratio detection in cells).

 

Today I will focus on methods to visualize actin in fixed or living cells – which belong to basic experimental set-ups in Cell Biology.

Actin staining of fixed cells

Actistain photo

Fig. 2: Swiss 3T3 fibroblasts stained with ActiStain 488 (green), Dapi (blue) and Anti Vinuclin (orange). Provided by Cytoskeleton Inc.

Often fluorescent phalloidins are used to stain actin in fixed cells. Phalloidin belongs to the group of phallotoxins produced by the mushroom Amanita phalloides (death cap mushroom). The natural toxicity of Phalloidin is due to its stabilizing effect on F actin in cells. Based on its affinity for F-actin and coupled to a fluorescent dye, it can be used to visualize F-actin.

Cytoskeleton Inc. offers a set of phalloidin based stains (Acti-Stains) coupled to a number of different fluorophores compatible with popular filter sets such as FITC, TRITC and Cy5. The stains are exceptionally bright and stable and are indeed offered at very economical prices compared to other phalloidin based stains coupled to fluorophores of similar stability.

Results of staining of Swiss 3T3 cells with ActiStain 488 are shown in Fig. 2.

Live-cell imaging of Actin

Live-cell imaging of actin has been quite tricky as far as actin labelling is concerned – either cells had to be transfected with vectors carrying the genetic information for fluorescently tagged actin or actin binding proteins or, labelled actin had to be micro-injected to single cells.

Actin

Fig. 3: 3D-SIM microscopy image of labeled Actin stress fibers in human primary dermal fibroblasts. Provided by Spirochrome.

Together with Spirochrome, tebu-bio launched in Europe the first tool to directly label actin in living cells with no need to transfect or micro-inject anything.

SiR-Actin is a cell permeable compound which stains F-actin in living cells. The stain is composed of a photostable silicon rhodamine-like (SiR) dye which can be used with standard Cy5 settings and a component (Jasplakinolide) which specifically binds to F-actin (Fig. 3). SiR-actin is compatible with Super-Resolution Microscopy like Stimulated Emission Depletion [STED] and Structured Illumination Microscopy [SIM].

f you would like to get an overview about the results SiR-actin users got so far, please have a look at my recent blog: User experience of SiR-Actin and SiR-Tubulin Live Cell Imaging.

 

 

 

Interested in our Phalloidin and/or SiR-based stains?

Leave your comment or request in the form below.

 

Anti cellulite compound evaluation with in vitro adipocyte-assays

Mechanisms leading to cellulite formation is complex. It involves lipid regulatory pathways and proinflammatory cross-talk that represent promising molecular targets in cosmetology. This post introduces a clever in vitro adipocyte-based assay targeting adipocytokines to better determine the anti cellulite effects of cosmetics compounds.

[Read more…]

Measuring total ubiquitinated proteins in cell lysates – made easy!

To date, detecting changes in the ubiquitylation of specific substrate proteins in response to external stimuli, e.g. stress, cytokine exposure, drug candidate treatment, etc., has been a long, labor-intensive process involving immunoprecipitation followed by gel electrophoresis and Western blot analysis.  This method is low through-put, resource intensive, and only semi-quantitative at best.

ELISA like method to measure all ubiquitinated proteins in cell lysates

Ubiquant S results 1

Fig. 1.: Decrease in LDLR ubiquitylation following treatment of transfected cells with an IDOL inhibitor.

To enable researchers to avoid the above mentioned limitations, LifeSensors has developed the UbiQuantTM S kit as a facile, robust, and quantitative alternative to IP/WB analysis. It is built on LifeSensors UbiQuant platform, in which ubiquitinated proteins in cell lysates are captured in the wells of a precoated microtiter plate using a proprietary ubiquitin binding reagent and then detected by an antibody against the protein of interest (either native or tagged) to quantitate the amount of the protein bound. Another version of this platform – the Ubiquitin Ubiquant ELISA – opens the possibility to measure the concentration of total free ubiquitin (poly- mono-) in biological samples including cell lysates, tissue homogenates, and plasma.

Fig. 1 shows a typical experiment using the UbiquantTM S assay. Decrease in LDLR ubiquitination following treatment of transfected cells with an IDOL (E3 ubiquitin ligase) inhibitor.  Cells are treated with different concentrations of an inhibitor of the E3 ubiquitin ligase IDOL and subsequently the decreasing ubiquitination of Low density lipoprotein receptor (LDLR) is measured with the UbiquantTM S assay. More detailed results can be found in an application note recently relased by LifeSenors.

Please note that if you want to order and use this kit and intend to use a tagged-substrate, you will have to specify the epitope tag in order to receive the appropriate antibody for detection with the kit(e.g. myc, HA, FLAG®, V5, GST, etc.  Unfortunately, His6 cannot be used for this assay.) Of course you can also use your own antibodies directed against your protein of interest.

Interested to apply this method to detect the total amount of ubiquitinated proteins in your sample? Get in touch through the form below, for advice on selecting the correct anti-tag antibody you might need.

Direct localization of MMP activity in 3D tumor invasion model

Recently, the non-FRET EnSens technology was launched for  in vitro assessment of specific protease activities (Enzium, Inc.). Up to date, the EnSens substates were validated for the detection of protease activity in microplate assays. Now, Enzium has extended the applicability of their Ensens method to 3 D live-cell imaging. On the top of this, they announce that the experimental procedure is easy and non-toxic for cell cultures and co-cultures. So how does live-cell imaging help you locate protease activities in in vitro tumor invasion assays ?

[Read more…]

3D monitoring of cell movement through Collagen I

The transition from non-invasive phenotype to invasive phenotype of tumor cells marks the switch from a benign tumor to a more malignant form of cancer. Understanding the mechanisms underlying this hallmark event, which enables tumor cells to invade through Extracellular matrix, is critical for discovering pathways and new targets to develop anti-metastatic strategies. In a previous post, A 96-well Invasion Assay Compatible with High Content Screening, I introduced a technology called Oris™ assays, which employs exclusion zone technology to facilitate unambiguous monitoring of cell migration from the periphery into a central circular detection zone. This assay is a high-throughput assay, but compatible with adherent cells only.  Today I’d like to introduce a new 3D Embedded Invasion Assay, compatible with both non-adherent and adherent cells.

[Read more…]

Modulating or inhibiting Caspase activities

In a previous post, I discussed Caspases as pharmaceutical targets – how to screen for inhibitors?

Today I would like to concentrate on Caspase inhibitors/modulators, which allow for in-depth characterisation of your enzyme of interest and which can serve as reference compounds in caspase inhibitor screenings. A short recap: Caspases (Cysteine-dependent aspartate-directed proteases) belong to the family of cysteine proteases and are involved in networks controlling cell death (apoptosis and necrosis) and inflammation. Amongst the 12 known human caspases, 5 have been described as playing a crucial role in  apoptosis (Caspase-3, -6, -7, -8, and -9), 4 have been linked to processes in inflammation (Caspase-1, -4, -5, and -12), and 3 (Caspase-2, -10, and -14) could not yet be exactly classified concerning their functions. [Read more…]

Differentiate between Apoptosis, Necroptosis, Autophagy & Ferroptosis

Are you looking for “Get your ELISA results before lunch! ” by Ana Arratzio? click here

Cell death can be caused by external factors such as infection or trauma – a process which is call necrosis. On the other hand, cell death can be mediated by intracellular programs – in this cases we talk about programmed cell death.

What are the different types of programmed cell death?

When we talk about programmed cell death we usually think of…

Apoptosis

Fotolia_71744424_XSThe induction of apoptosis leads to characteristic cell changes and finally to death (see How to measure early apoptotic events). These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. Furthermore, changes affecting the membrane, nucleus, cytoplasm, and mitochondria occur. Apoptosis involves a complex cascade of reactions regulated by specific proteases called caspases (see Caspases as pharmaceutical targets – how to screen for inhibitors?), and results in DNA degradation. Apoptotic processes have been researched in an extensive variety of diseases. Excessive apoptosis causes atrophy, whereas an insufficient amount results in uncontrolled cell proliferation, such as cancer.

Another quite prominent type of programmed cell death is represented by…

Autophagy

Autophagy process - Blog ThumbnailAutophagy describes the fundamental catabolic mechanism during which cells degrade dysfunctional and unnecessary cellular components (see How to manipulate and measure Autophagy). This process is driven by the action of lysosomes and promotes survival during starvation periods, as the cellular energy level can thus be maintained. During autophagy autophagosomes are generated, organelles which are surrounded by double membranes and which contain the cellular components to be degraded. The autophagosome formation is induced by class 3 phosphoinositide-3-kinase, Atg 6 (autophagy-related gene 6) and ubiquitin or ubiquitin-like modifications of the target proteins. Subsequently, autophagosomes traffick through the cytosol of the cell and finally fuse with lysosomes to form an autolysome. After this fusion the cargo is degraded by lysosomal hydrolases. Excessive autophagy leads  to cell death – a process which can be morphologically differentiated from apoptosis.

But there are less known types of programmed cell death, such as…

FerroptosisFerrostatin-0 - Andrea 11

This process is triggered by an iron-dependent accumulation of lethal ROS in cells. It can be induced by e.g. erastin which blocks the cellular uptake of cystine and thus blocking the intracellular antioxidant defense mechanism by limiting the production of intracellular glutathione (GSH), the primary cellular antioxid. ROS generation is iron-dependent as its accumulation and cell death can be supressed by the iron chelator deferoxamine (see Ferroptosis – and the way to inhibit it).

And finally…

Necroptosis

Necroptosis is indeed a programmed form of necrosis. As in all forms of necrotic processes, cells break open and leak their contents into the intercellular environment. In contrast to necrosis, leaking of the membrane during necroptosis is regulated by the cell. Necroptosis has been well described as a non-apoptotic “cellular suicide” process which represents a viral defense mechanism. Moreover, it plays a role in inflammatory diseases such as pancreatitis and Crohn’s disease.

Tools to differentiate between Apoptosis, Necroptosis, Autophagy, and Ferroptosis

Focus Biomolecules recently launched a set of compounds which are know to selectively inhibit one of the four programmed cell death types described above.

If you are interested to confirm a hypothesis that cells are undergoing a certain response due to the “hypothesized” mechanism – these inhibitors can be of great help to you!

Ferrostatin 1 - structure

Ferrostatin-1

  • Ferrostatin-1  – a specific inhibitor of ferroptosis. Ferrostain-1 has been shown to control lipid ROS (reactive oxygen species)
  • 3-Methyladenine – a specific autophagy inhibitor
  • Necrostatin-1 – a specific inhibitor of necroptosis
  • Z-VAD-FMK – one of a series of well defined apoptosis modulators (you’ll find here an overview about apoptosis modulators)

Any questions or comments? Please use the form below!

 

 

Ferroptosis – and the way to inhibit it

A lot of compounds are known and available to modulate (inhibit or activate) the most prominent mode of programmed cell death – Apoptosis. In a recent post, I summarized methods to detect early Apoptosis and at the end of this post you’ll find a list of apoptosis modulators (available through tebu-bio). But there are other modes of programmed cell death, such as Autophagy (see How to manipulate and measure Autophagy), Necroptosis, and Ferroptosis.

Today I would like to focus on Ferroptosis, and invite you to take a look at the first Ferroptosis inhibitor on the market. [Read more…]