How to measure early apoptotic events?

Apoptosis and cellular apoptotic events. Source: tebu-bio

Fig. 1: Process of Apoptosis

Apoptosis is the most prominent process of programmed cell death (for an overview see Fig. 1). Biochemical events lead to characteristic cell changes and death. 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 (take a look at previous posts on 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.

Besides apoptosis other types of programed cell death are known, such as autophagy (see How to manipulate and measure Autophagy?), necroptosis, and ferroptosis (look out for an imminent post I’ll be doing about this iron-dependent form of cell death very shortly, as well as tools to differentiate between apoptois, necroptosis, autophagy, and ferroptosis).

In this post, let’s take a look at methods and kits allowing to measure early apoptotic events. [Read more…]

How to detect active caspase in cells?

In a recent post I gave an overview about the role of Caspases in different human diseases and introduced tools to measure Caspase activity and inhibition by compounds in homogenous biochemical assays: Caspases as pharmaceutical targets – how to screen for inhibitors? Today I would like to give you an overview about a method and kits which allow to detect active Caspases in cells and which give an insight into the apoptotic status of the respective cells. Hence the effects of inducers of apoptosis can be investigated in living cells. [Read more…]

Staining Actin and Tubulin – from WB to Live Cell Imaging

Anti Anti Ab - IF photo

Fig. 1: Immunofluorescence images of mouse Swiss 3T3 cells stained with anti-actin antibody (027AAN01).

Actin and Tubulin, as the major cytoskeleton structures, are crucial components of a plethora of processes in cell biology. Both are very much involved in stabilizing the cell shape, and especially in cell movements (e.g. cell migration) and intracellular movements and transport mechanisms.

Thus visualizing Actin and Tubulin in fixed or living cells and detecting them in biological samples (e.g. in Western Blots) belongs to basic experimental set-ups in Cell Biology.

A broad range of tools is available for all kind of experimental levels, in this post we’ll take a look at some of these you can use from Western Blot to Live Cell Imaging. [Read more…]

User experience of SiR-Actin and SiR-Tubulin Live Cell Imaging

Very recently we launched new Live Cell Imaging tools: SiR-Actin and SiR-Tubulin, produced by Spirochrome.

These stains allow you to stain actin and tubulin in living cells without the need to transfect cells – as I described in my previous posts on these tools:

Today, I invite you to take a look at the brilliant results users of the stains have obtained. Some of them have already been published during the past months.

Most recent publications:

One of the most recent publications using SiR-Actin comes from the lab of the Nobel price winner Stefan W. Hell, who is one of the directors of the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany:

STED Nanoscopy Reveals the Ubiquity of Subcortical Cytoskeleton Periodicity in Living Neurons, Elisa D’Este, Dirk Kamin, Fabian Göttfert, Ahmed El-Hady, and Stefan W. Hell, Cell Reports, 10, 8, 1246–1251 (2015)

By using STED (stimulated emission depletion) microscopy, Hell’s group could visualize the periodic subcortical actin structure in axons and dendrites in cultured hippocampal neurons. These results were obtained by staining Actin with SiR-Actin.

Another recent paper even brought an image of HEK293 cells co-expressing CaVβ and CaV1.2 L-type calcium channel and stained for actin filaments using SiR-Actin straight to the front cover of the respective JBC issue.

Direct Interaction of CaVβ with Actin Up-regulates L-type Calcium Currents in HL-1 Cardiomyocytes, Gabriel Stölting, Regina Campos de OliveiraRaul E. Guzman, Erick Miranda-Laferte, Rachel Conrad, Nadine Jordan, Silke Schmidt, Johnny Hendriks, Thomas Gensch,  and Patricia Hidalgo, Journal of Biological Chemistry, 290, 4561-4572 (2015)

The group around Patricia Hidalgo at the Institute of Complex Systems in Juelich, Germany, could show that the β-subunit (CaVβ) n of cardiac L-type calcium channels associates directly with actin filaments – again SiR-Actin was successfully used to get these results.

Videos showing the use of SiR-Tubulin

As SiR-stains are very photo stable and do not show toxic effects, even in long-term incubation, they are excellent tools for visualizing biological processes over time in videos.

We show here an example of dividing HeLa cell expressing mcherry-H2B (red) stained with SiR-Tubulin (green). Data have been collected by confocal imaging (Courtesy of Daniel Gerlich and Claudia Blaukopf, Institute of Molecular Biotechnology, Vienna, Austria).

Another video shows newborn mouse primary cardiac myocytes stained with SiR-actin. The authors used high speed (50fps) confocal imaging (Courtesy of Adam Kwiatkowski and Simon Watkins, Department of cell biology and center for biologic imaging, University of Pittsburgh, US).

Results provided by our customersHUVEC monolayer - Erik T. Valent and Geerten P. van Nieuw Amerongen - Amsterdam

HUVEC monolayer, stained with SiR-Actin. A ZEISS Axiovert 200 Marianas inverted microscope with custom ZEISS 40x air lens was used (Courtesy of Erik T. Valent and Geerten P. van Nieuw Amerongen, VU Medisch Centrum, Amsterdam , The Netherlands).

ACTIN MOD_J_Millan_2015_1Human endothelial cells B4G12 were grown on Ibidi® µ-Slide 4 Well dishes at confluence. Cells were labeled for 2h at 37°C/5% CO2 with 0.2 mM SiR-Actin and a confocal image was acquired exciting with a Laser Helio Neon of 637 nm with a Zeiss Confocal LSM510 META system (Courtesy of Cristina Ortega Muñoz and Jaime Millan, Centro de Biologica Molecular, Madrid, Spain).

 

SiR-Actin staining for transient labeling of breast cancer cells implanted into a xenogeneic zebrafish host. SiR-Actin stained cells (MDA-mb231B1 dsRED), stained overnight, followed for 6 days via confocal imaging (Leica TCS SPE) microscope (63x objective). Note the retention of SiR-Actin in vitro, and the absence of cellular artefacts with concentrations of SiR-Actin below 100 nM. Cytosolic CMV driven dsRED shown in red and Sir-Actin shown in cyan (Courtesy of Arwin Groenewoud and B. Ewa Snaar-Jagalska, Institute of Biology, Leiden, The Netherlands).

Sir-Actin (SC001)staining for transient labeling of breast cancer cells implanted into a xenogeneic zebrafish host - Arwin Groenewoud and B. Ewa Snaar-Jagalska, Leiden

STORM spines (1)Primary neurons derived from cortex + hippocampus of wild type mouse C57BL/6J were cultured for 19 days and subsequently stained with SiR-Actin and visualized with a Nikon NSTORM to shown spine formation (Courtesy of Oxana Klementieva, Gunnar Gouras (Lund University) and Catherine Kitts from Lund University Bioimaging Center [LBIC], Sweden).

We would like to thank all the users who provided the pictures and results!

And of course, we invite all researchers who would like to test the SiR stains in their laboratory to contact us through the form sheet below.

First Pin1 Alzheimer research assay on the market

Pin1 (Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1; Peptidyl-prolyl isomerase, PPIase) is an enzyme that isomerizes phospho/Threonine-Proline motifs. Upon binding (to various target proteins) it can function as a protein regulator as it plays a role in post phosphorylation control.

Deregulation of PIN1 can lead to various diseases. Up-regulation is discussed to be implicated in specific cancer types, and down-regulation may be implicated in the pathogenesis Azheimer’s disease (1). [Read more…]

Post-translational modifications regulate Ral GTPases

RalA and RalB GTPases regulate cell motility, morphology, signaling, vesicular trafficking, and endo/exocytosis. The regulation of these functions is critical for the development and spread of cancer, implicating Ral in oncogenesis and metastasis. Both isoforms are integral for Ras-mediated tumorigenesis, metastasis, and invasion. Despite sharing 82% amino acid sequence identity, effectors, and structural/biochemical properties, RalA and RalB have their own unique functions in oncogenesis due to distinct subcellular localization and differential effector interactions. Ral localization, binding partners, and function are regulated by post-translational modifications (PTMs).

print_logoIn their recent newsletter, Cytoskeleton Inc. summarize recent findings about the relevance of geranylgeranylation, carboxymethylation, palmitoylation, phosphorylation, and ubiquitination in regulating Ral activity, subcellular localization, effector binding, and ultimately, function.

You can download a copy of this newsletter, or if you have any questions or comments, don’t hesitate to get in contact through the form below.

Kits to measure RalA activation

If you’d like to get an overview about what’s available in the small G protein field, take a look at this Small GTPase product guide.

How to manipulate and measure Autophagy?

The term Autophagy was introduced by Christian de Duve during the Ciba Foundation Symposium on Lysosomes – which was held in London in February 1963. In 1974 he was honoured with the Nobel price in Physiology or Medicine for his pioneering research about peroxisomes and lysosomes.

In this post, I’d like to give you an overview of autophagy and its implication in cell biology, and tools to manipulate and detect autophagy in cells. [Read more…]

How to measure Glycosaminoglycans and Proteoglycans?

Glycosaminoglycans (or mucopolysaccharides) are long un-branched polysaccherides which consist of disaccharide repeats. The repeats usually (with the exception of Keratan sulfate) consist of on amino sugar (N-acetylgalactosamine of N-acetylglucosamine) and a uronic sugar (iduronic acid or glucuronic acid) or galactose.

Glycosaminoglycans are highly polar and function as water attractants, thus they are useful as shock absorbers or lubricants.

Four glycosaminoglycan classes can be distinguished:

Structure chondroitin sulfate

Chrondroitin sulfate

  • Heparin/heparan sulfate
  • Chondroitin sulfate/dermatan sulfate
  • Keratan sulfate
  • Hyaluronic acid

Dermatan sulfate

 

 

 

 

 

These structures differ in their core disaccaride structure and their synthesis pathways. While Heparin/heparan sulfate, Chondroitin sulfate/dermatan sulfate and Keratan sulfate are produced by the Golgi apparatus, Hyaluronic acid is synthezised by integral membrane synthases which immediately secrete the elongated disaccharide chain.

Proteoglycans – core proteins attached to glycosaminoglycans

Proteoglycans consist of core proteins which are covalently linked to glycosaminoglycans – either mono- or poly-glycosylated. The glycosaminoglycan is coupled though a tetrasaccharide bridge to a Serin residue of the protein. Proteoglycans represent the major component of the animal extracellular matrix where they form complex networks with other proteoglycans and fibrous matrix proteins such as collagen. These networks are involved in binding water and diverse cations.

Mucopolysaccharidoses are genetic discorders which lead to the inability to degrade proteoglycans. As a consequence an accumulation of proteoglycans occurs which subsequently leads to a number of pathological symptoms (dependent on the type of proteoglycan which cannot be degraded).

One assay to measure almost all glycosaminoglycans and peptidoglycans

Flowchart Blyscan

Fig. 1: Blyscan Assay Flowchart

The Blyscan Assay (see flowchart Fig. 1) is a quantitative dye-binding method for the analysis of sulfated proteoglycans and glycosaminoglycans. Test material can be assayed directly when present in a soluble form, or following papain extraction from biological materials. The assay can be used to measure the total glycosaminoglycans content and can also be adopted to determine the O- and N-sulfated glycosaminoglycan ratio within test samples.
The dye label used in the assay is 1, 9-dimethylmethylene blue and the dye is employed under conditions that provide a specific label for the sulfated polysaccharide component of proteoglycans or the protein free sulfated glycosaminoglycan chains.

Note: the assay is not suitable for small sulfated disaccharide fragments or for samples containing alginates, as these contain uronic acid.

Results Blyscan

Fig. 2: Recovery of soluble glucosaminoglycans from mouse tissues, with 3 and 18 hours of hot papain.

For analysing which soluble extracts?

  • fibrous and hyaline cartilages
  • arteries, heart, lung, skin and other material containing extracellular matrix, (connective tissue) and solid tumours (results see Fig. 2)
  • extracellular matrix components that may be released by live cells into the culture medium, some of which can be attached to cell culture plasticware
  • soluble glycosaminoglycans from synovial fluid, urine and gel chromatography fraction aliquots

Would the Blyscan assay be of use in your research? Share your comments or questions below!

 

 

 

Proteasome and (de)ubiquitination enzyme inhibition

In 2014, the U.S. Food and Drug Administration (FDA) approved the proteasome inhibiting drug Velcade (Bortezomib) for the retreatment of adult patients with Multiple Myeloma. My colleague Philippe Fixe discussed the use of Bortezomib in his post Proteasome inhibitor approved by FDA for Myeloma retreatment.

In this post, I’d like to give you an overview of the compounds which can be used to inhibit proteasome activity and furthermore the activity of De-ubiquitinases (DUB, Isopeptidases), enzymes which catalyze the de-ubiquitination of ubiquitinated proteins and which are seen as very promising drug targets especially in cancer drug development. [Read more…]

TUBEs – efficiently detect & purify poly-ubiquitinated proteins

Traditional methods to detect and purify poly-ubiquitinated proteins require either anti ubiquitin antibodies or Ubiquitin Binding Associated domains (UBAs) which display rather low affinity for Ubiquitin and show only little (if any) specificity for specific ubiquitin linkages (e.g. K63 or K48). Furthermore, these strategies require the inclusion of inhibitors of both Deubiquinating enzymes (DUBs) and Proteasome activity to protect the integrity of poly-ubiquitylated proteins, which might alter cell physiology, which in turn may negatively impact the result or introduce experimental artifacts.

To overcome these problems, LifeSensors have developed Tandem Ubiquitin Binding Entities (TUBEs). [Read more…]