10. S. W. Tait, D. R. Green, Nat. Rev. Mol. Cell Biol.11, 621–632 (2010).
This review explores the relationship between the mitochondria and apoptosis with a focus on mitochondrial outer membrane permeabilization and the factors that coordinate the process.
8. J. B. Chang, J. E. Ferrell Jr., Nature 500, 603–607 (2013).
This paper establishes that Cdk1-mediated trigger waves help to successfully coordinate mitosis over long distances in large cells like that of the Xenopus laevis. Cdk1 is a protein that facilitates the onset of mitosis in the cell cycle.
9. J. B. Chang, J. E. Ferrell Jr., Cold Spring Harb. Protoc. 10.1101/ pdb.prot097212 (2018).
This is a methods paper which describes an experimental system that promotes robust cell cycling in Xenopus laevis egg extracts. The authors find that placing frog extracts in Teflon tubes enables robust and better cycling compared to other materials.
L. Gelens, G. A. Anderson, J. E. Ferrell Jr., Mol. Biol. Cell 25, 3486–3493 (2014).
This paper examined the different types of trigger waves and showed how they arise
P. Deming, S. Kornbluth, Methods Mol. Biol. 322, 379–393 (2006).
This paper demonstrated that molecular events in the apoptosis pathway can separately studied using cytosolic extracts isolated from the crude Xenopus egg cytoplasmic extracts.
D. D. Newmeyer, D. M. Farschon, J. C. Reed, Cell 79, 353–364 (1994)
This paper demonstrated four important things: (a) The Xenopus egg extract can be used as a model for studying apoptosis. (b) Exogenous nuclei can be added to the extract to monitor apoptosis. (c) Exogenous Bcl-2 can be used to block apoptosis in the Xenopus egg extracts. (d) Apoptosis in Xenopus egg extracts is dependent on enriched mitochondria in the extracts.
Imaging studies on mammalian cell lines (18–20), cardiac myotubes (21, 22), and syncytial human trophoblasts (23) have shown that apoptosis typically initiates at a single discrete focus or a small number of discrete foci and then spreads rapidly throughout the cell, and in some of these studies the propagation velocities appeared to be constant over distances of ~100 μm
Scientists have clearly shown that apoptosis takes place in different cell types and beginning at specific points/positions, it spreads through apoptotic cells at a constant speed.
This paper was able to demonstrate that the spreading of apoptosis through cells occurs via trigger waves.
The x2-versus-t relationship was linear for at least an hour (fig. S4C), consistent with random walk diffusion. Thus, over this time scale and this distance scale, we found no evidence for a trigger wave
In the absence of the mitochondria, the authors found that the spread of apoptosis signals were not consistent with propagation by trigger waves.
We fractionated a crude cytoplasmic extract (2, 3) (fig. S3A) and verified by immunoblotting that the resulting cytosol was largely devoid of mitochondria, as indicated by the absence of a mitochondrial marker protein, the voltage-dependent anion channel (fig. S3B).
Here, the authors wanted to test how the speed of apoptotic trigger waves are affected in the absence of the mitochondria. As a result, they obtained the cytosolic extract by fractionation and checked for the absence of the mitochondria using a marker protein by immunoblotting.
The marker protein resides and functions in the mitochondria and hence the absence of this protein can be used as an indicator for the absence of the mitochondria in the extract.
Immunoblotting (also called western blotting) is a technique used to detect the presence and levels of a specific protein in biological samples.
Here is a video illustrating how immunoblotting is performed in the lab: https://www.youtube.com/watch?v=OkH8u84t84M
Bak and Bax proteins
These two proteins activate apoptosis by piercing and creating pores within the outer membrane of the mitochondria and causing the release of cytochrome c. In an interesting research, scientists also found that both proteins interact to trigger inflammation by the release of mitochondrial DNA. This new finding may provide avenues to manage inflammatory responses in cancer patients during treatment.
Read more in the ScienceDaily: https://www.sciencedaily.com/releases/2022/02/220204113438.htm
reconstituted nuclei
Used here to refer to the sperm chromatin added to the extract
amplitude
The maximum amount of distance a wave can travel from its rest position
determine whether apoptosis spread up the thin tube in a diffusive fashion, with propagation slowing as apoptosis proceeded, or at a constant velocity as expected for trigger waves
The hypothesis here is that: a diffusive apoptotic wave will slow down over the course of time while apoptosis propagated by trigger waves will maintain a steady speed during progression.
The powerful biochemical approaches provided by the Xenopus system have made Xenopus eggs and extracts useful model systems for the study of apoptosis
Scientists have shown that the Xenopus egg extract is an effective medium for studying the process of apoptosis.
Every egg that showed a wave had increased caspase activity (Fig. 4, E and F, red symbols). We also collected eggs during the same time period that had not displayed a wave. None of these eggs had increased caspase activity
The authors detected caspase activity in eggs that displayed wave propagation under apoptotic conditions and did not detect any caspase activity in eggs that did not have any wave propagation.
This evidence indicated that caspase activation is involved in the propagation of apoptotic waves in intact eggs.
The TMRE wave propagated at the same speed as the caspase trigger wave reported by Z-DEVD-R110 in the same tube (38 μm/min)
Here, the authors confirmed that TMRE (which measures mitochondrial membrane potential) can be used to monitor and assess apoptotic trigger waves.
This video shows the propagation of apoptosis through a frog egg extract as visualized using the fluorescent dye TMRE: https://www.youtube.com/watch?v=3rIS7Y4LB6U
As shown in movie S1 and Fig. 1, B and C, apoptosis progressed up the thin tube at a constant speed of 27 μm/min over a distance of several millimeters. In five independent experiments, apoptosis always propagated linearly, without showing any signs of slowing down or diminishing, and the average speed was 29 ± 2 μm/min (mean ± SD). In contrast, the 10-kDa dye spread only a few hundred micrometers (Fig. 1B), implying that neither simple diffusion nor any unintended mixing could account for the spread of apoptosis.
The goal here was to find out if diffusion was responsible for the spread of apoptotic signals. Because diffusive spread is known to slow down over time, its was used by the author's as a mark to determine if the spread of apoptosis was by diffusion.
Results showed that apoptosis spread at a constant speed and did not slow down indicating that diffusion was not responsible.
To further test this possibility, we added recombinant GST–Bcl-2 protein to cytoplasmic extracts to see whether trigger waves were affected. Bcl-2 is a stoichiometric inhibitor of the pro-apoptotic truncated Bid (tBid) protein and of the pore-forming Bak and Bax proteins, and so the expectation was that GST–Bcl-2 would slow the trigger waves
Here, the authors wanted to know if Bax and Bak contributed to the generation and speed of apoptotic trigger waves.
To test this they blocked the activity of Bax and Bak by adding Bcl-2 (known inhibitor of Bax and Bak) to the extract and tested the speed of the apoptotic trigger waves as before.
fluorogenic
Something that generates fluorescence.
Example: The green fluorescent protein (GFP) is a protein that emits bright green fluorescence upon exposure to light in the blue to ultraviolet range. Hence GFP can be said to be fluorogenic.
cytosolic proteins
Large complexes of enzymes in the cytosol of the cell that are involved in various cellular pathways including metabolism, biosynthesis and cell signalling.
The cytosol is the aqueous part of the cytoplasm of a cell where cellular components (e.g., organelles) are suspended.
positive feedback loops
AP Biology Essential knowledge 2.C.1.b: Positive feedback mechanisms amplify responses and processes in biological organisms. The variable initiating the response is moved farther away from the initial set-point. Amplification occurs when the stimulus is further activated which, in turn, initiates an additional response that produces system change.
Link https://secure-media.collegeboard.org/digitalServices/pdf/ap/10b_2727_AP_Biology_CF_WEB_110128.pdf
kymograph
Distance-time plots that are used in biology to monitor and track the movement of fluorescent or fluorescently-labeled particles (e.g., molecules, organelles etc.) along a predicted path.
bistable biochemical reactions
Describes a system with two stable equilibrium states and therefore capable of resting in (or reaching) either of the two states. It arises from feedback loops that exhibit mutual activation or mutual repression.
cell cycle
AP Biology Essential knowledge 3.A.2.a: The cell cycle is a complex set of stages that is highly regulated with checkpoints, which determine the ultimate fate of the cell.
Link: https://secure-media.collegeboard.org/digitalServices/pdf/ap/10b_2727_AP_Biology_CF_WEB_110128.pdf
interphase
Essential knowledge 3.A.2.a.1: Interphase consists of three phases: growth, synthesis of DNA, preparation for mitosis.
Link: https://secure-media.collegeboard.org/digitalServices/pdf/ap/10b_2727_AP_Biology_CF_WEB_110128.pdf
chimeric protein
A protein made by combining two or more genes that code for the synthesis of their individual proteins joined together. Synthesis results in the formation of one protein with specific functional properties.
cytoplasmic extract
Contains the all the internal contents of the cell
cell-free Xenopus laevis egg extracts
Preparation obtained by crushing the eggs of the African clawed frog (Xenopus laevis) to release its internal contents.
glutathione S-transferase
A protein that is popularly used as a tag for the purification of recombinant proteins. It can be fused to either ends of the desired protein, usually the end that does not affect the function of the target protein.
A recombinant protein is produced by cloning a gene into a system that allows the expression of that gene and the translation of its gene product.
self-regenerating trigger waves
Self-renewing biological phenomenon that is capable of long-range transmission/propagation of information.
Apoptosis
A series of events that are genetically programmed to result in the death of a cell. It takes place as a normal part of the growth and development of an organism.
Here is an animation video that illustrates apoptosis: https://www.youtube.com/watch?v=-vmtK-bAC5E
Here is an introductory lecture to the process of apoptosis https://www.youtube.com/watch?v=31dmXwBZNWI&ab_channel=iBiology
green fluorescent protein
A protein that emits bright green fluorescence upon exposure to light in the blue to ultraviolet range.
Fluorescence occurs when light of a shorter/high-energy wavelength (such as UV light) is directed on a surface allowing a specific component (e.g. green fluorescent protein) on the targeted surface to emit light of a specific wavelength.
calcium waves
An increase in the concentration calcium ions in the cytosol which causes a succession of the same events to take place in a wave-like manner. The generation of calcium waves can therefore be viewed a positive feedback mechanism.
Here is a visualization of the propagation of calcium waves in a fertilized egg: https://www.youtube.com/watch?v=M8GC-zwlF8w
These experiments implicate caspase-3 and/or -7 as well as Bcl-2 in the regulation of the apoptotic trigger waves. The experiments also show that the trigger waves are relatively robust; they are still present, though with reduced speeds, in extracts depleted of mitochondria or treated with maximal doses of GST–Bcl-2 or Ac-DEVD-CHO
Together, the results presented in Figure 3 indicate that caspase (-3, -7) and pro-apoptotic proteins (Bax and Bak) activity are essential in dictating the speed of apoptotic trigger waves in Xenopus egg extracts
reconstituted extract
Used here to refer to the cytosolic extract where the mitochondria has been added back.
Remember that the cytosolic extract is devoid of mitochondria.
fluorophore
A fluorescent compound that can re-emit light upon excitation. They are typically used in techniques such as fluorescent imaging.
time-lapse fluorescence microscopy
A form of microscopy where microscopic images are first recorded sequentially and then viewed at a higher speed to provide an accelerated view of the process being visualized.
nuclear localization sequence
A short protein sequence that serves as a signal for target proteins to be transported from the cytoplasm to the nucleus. In this chimeric (fusion) protein, the sequence the GST-GFP component to be transported into the nucleus.
allele
One of two or more versions of a gene (found at the same place on the chromosome) that comes about due to changes in the DNA sequence.
Fractionation
A method used to separate cellular components
Adding GST–Bcl-2 decreased the wave speed (Fig. 3, A and B, and movie S5). The maximum effect [at 400 nM added GST–Bcl-2, which compares to the estimated endogenous Bcl-2 concentration of approximately 140 nM (14)] was a reduction of the speed to about 13 μm/min, the speed seen in purified cytosol. Added GST–Bcl-2 had no effect on the trigger wave speed in cytosolic extracts (Fig. 3, C and D), which emphasizes that the waves seen in purified cytosol are probably not caused by contaminating mitochondria. GST–Bcl-2 decreased the trigger wave speed in reconstituted (cytosol plus mitochondria) extracts (Fig. 3, E and F), just as it did in cytoplasmic extracts
The authors found that blocking the activity of Bax and Bak (via Bcl-2) reduces the speed of apoptotic trigger waves in cytoplasmic and reconstituted cytosolic extracts (both of which have mitochondria) but does not have any effect on the relatively slower speed of apoptotic trigger waves in purified cytosolic extracts (extracts without mitochondria).
This evidence suggested that Bax and Bak positively contribute to the generation and speed of apoptotic trigger waves in extracts containing the mitochondria.
During this same time period, a surface wave often appeared, originating near the vegetal pole and propagating toward the animal pole, with a typical apparent speed of ~30 μm/min
After treating eggs under apoptotic-inducing conditions, the authors observed the movement of a wave (with a speed characteristic of the speed of apoptotic trigger waves seen in the extracts) on the surface of the egg from one end of the egg (called vegetal pole) to the other end of the egg (called animal pole)
The vegetal pole is the portion of an ovum (mature female reproductive cell) containing most of the yolk and little cytoplasm while the animal pole is the portion of an ovum that contains the nucleus and less yolk.
The vegetal pole and the animal pole are opposite each other on the ovum.
These findings support the idea that spontaneous apoptosis typically initiates near the vegetal pole of the egg and propagates outward and upward from there as a ~30-μm/min trigger wave
In summary, the evidence presented in Figure 4 demonstrated that apoptotic trigger waves were also propagated in intact oocytes and eggs.
The surface waves propagated at an apparent speed of ~30 μm/min, similar to the wave speeds seen in apoptotic extracts. Control oocytes injected with Texas Red–dextran in water did not exhibit these surface waves. These findings indicate that apoptotic trigger waves can be produced in oocytes
The authors visualized apoptotic waves in intact oocytes (immature eggs) of Xenopus laevis suggesting that apoptotic trigger waves occur in oocytes.
Both reporters showed that the wave speed decreased as the inhibitor concentration increased
The authors observed that increasing the concentration of the caspase inhibitor in the extract resulted in decreasing speed of the apoptotic trigger waves.
As a result, the higher the concentration of the caspase inhibitor, the lower the speed of the apoptotic trigger waves.
This evidence indicated that caspase-3 and caspase-7 contribute positively to the speed of apoptotic trigger waves.
If the MOMP–caspase–BH3 protein loop contributed to the generation of trigger waves, inhibition of the executioner caspases would be expected to slow or block the waves (Fig. 1A). To test this, we added the caspase-3 and -7 inhibitor N-acetyl–Asp-Glu-Val-Asp–aldehyde (Ac-DEVD-CHO) to the reconstituted extracts. Because high concentrations of the inhibitor make it difficult to monitor trigger waves with the fluorogenic caspase substrate Z-DEVD-R110, we used an additional probe, tetramethylrhodamine ethyl ester (TMRE), a red fluorescent dye that responds to changes in mitochondrial membrane potential
Here, the authors inhibited caspase activity in reconstituted cytoplasmic extracts and tested its effect on the speed of apoptotic trigger waves by monitoring the caspase activity and mitochondrial membrane potential (mark of an active mitochondria).
BH3 protein
Proteins that possess a characteristic short protein (or peptide) sequence termed the "BH3 domain" (also called the death domain).
Members of this family of proteins are known to trigger key mitochondrial events that kill the cell and hence are associated with apoptosis.
However, in experiments with either cytoplasmic extracts or reconstituted extracts, more than half of the time (in 12 of 22 or 21 of 37 tubes, respectively) a second spontaneous apoptotic wave emerged elsewhere in the tube
Here, the authors found that mitochondria-containing apoptotic (that is cytoplasmic or reconstituted) extracts sometimes generated a second apoptotic wave somewhere in the tube in addition to the first wave which originates from the bottom of the tube.
we determined the trigger wave speed to be half maximal at a mitochondrial concentration of 1.3 ± 0.6% (mean ± SE), which is estimated to be ~40% of the physiological mitochondrial concentration in Xenopus eggs. Thus, an average concentration of mitochondria is sufficient to generate apoptotic trigger waves of near-maximal speed, and the wave speed would be expected to drop in mitochondrion-poor regions of the cytoplasm
The authors found that only an average concentration of mitochondria is required to produce apoptotic trigger waves at their maximum speed.
As was the case in Fig. 2B, the speed of the wave front fell during the first ~120 min, consistent with diffusive propagation, but once the speed reached ~14 μm/min it remained constant for many hours (Fig. 2E). This suggests that purified cytosol is capable of generating apoptotic trigger waves, albeit with a substantially lower speed than that seen in cytoplasm or in cytosol supplemented with mitochondria
Here, the authors found that the purified cytosol (without mitochondria) produces slower apoptotic trigger waves when compared to that of the reconstituted cytosol (with mitochondria).
This evidence indicates the mitochondria positively contributes to the speed of apoptotic trigger waves.
curve fitting
The process of constructing a curve that has the best fit to a series of data points.
Moreover, the reconstitution restored the trigger waves (movie S3 and Fig. 2D). The propagation distance increased linearly with time (Fig. 2D), as it did in crude cytoplasmic extracts (Fig. 1), and propagation occurred at a constant speed of 39 μm/min, somewhat faster than that observed in cytoplasmic extracts. The signal propagated over a long distance (6000 μm) with little loss of amplitude and no loss of speed (Fig. 2D)
The authors found that the addition of the mitochondria caused apoptosis to spread through the extracts by trigger waves.
This evidence showed that the mitochondria is required for the propagation of apoptotic signals via trigger waves.
In agreement with previous reports (3, 13), the caspases were briskly activated (Fig. 2A). Thus, mitochondria are not essential for cytochrome c–induced activation of executioner caspases in Xenopus extracts
The authors found that caspase activity was not blocked in the absence of the mitochondria indicating that the mitochondria was not necessary for the activation of caspases by cytochrome c.
chromogenic caspase assay
An experiment which uses colored of fluorescent substrates (e.g., Z-DEVD-R110) to quantify the activity of an enzyme (e.g., caspase).
As a result, the higher the fluorescence detected, the higher the enzyme activity.
mitochondrial outer membrane permeabilization (MOMP)
Process where pores are created in the outer membrane of the mitochondria to facilitate the release of cytochrome c which in turn activates caspases necessary for apoptosis to occur.
XIAP
A protein that blocks apoptosis by keeping caspase-3 and caspase-7 in an inactive state where they are not able to perform their roles in apoptosis.
Fluorescence spread up the tube at a constant speed (in this experiment, 33 μm/min). In eight experiments, the average speed was 30 ± 3 μm/min (mean ± SD)
Using a second method, the authors confirmed that apoptosis spread through the extract at a constant speed exhibiting the properties of trigger waves
hydrolyzed
To break down (a compound) by chemical reaction with water.
substrate
A molecule upon which an enzyme (e.g., caspase) acts. Enzymes catalyze chemical reactions involving the substrate(s).
The apoptotic activity propagated from the induction terminus to the distal terminus at a constant speed of 32 μm/min, consistent with a self-sustaining process
Apoptosis is spread by trigger waves as indicated by the spread of the apoptosis waves/signals at a steady speed.
interphase extract
Refers to "interphase cytoplasmic extract" with no cycloheximide treatment and no cytochrome c.
chromatin
A nucleic acid–protein complex that makes up eukaryotic chromosomes
apoptotic extract
Refers to the cycloheximide-treated interphase with cytochrome c
cytochrome c
Small protein which is loosely associated with the inner membrane of the mitochondria [powerhouse of the cell]. Perturbations to the inner mitochondrial membrane causes the release of cytochrome c which then activates apoptosis.
Texas Red–conjugated dextran
Dye used to monitor cell division and track the movement of live cells.
cycloheximide
A chemical that blocks protein synthesis in eukaryotic organisms. Upon exposure, many cell types rapidly undergo apoptosis.
video microscopy
Provides live feed (real-time) video image directly to a computer for visualization and monitoring.
inflammatory response
Defense mechanism used by an organism to remove harmful agents (such as damaged cells and pathogens).
regulatory network
Used here to describe a system of biological molecules that interact to obtain desired outcome such as apoptosis.
embryonic
Relating to the embryo
meiosis II
A stage in cell division where chromatids [one half of a duplicated chromosome] separate.
metaphase
The second stage of cell division where the chromosomes prepare to be separated into daughter cells by aligning in the equator of the parent cell.
cell signaling
A form of cellular communication involving the reception, processing and transmission of biological and chemical signals.
caspase
Proteases (enzymes that break down proteins) that function as the main effectors of apoptosis
oocytes
An immature egg
inhibitor studies
Used here to describe assays or experiments involving the blocking of activity of an enzyme or biological process.
homeostasis
Steady state conditions (no disturbances)
evolutionarily conserved
Describes a process or feature that is maintained and takes place across different species (organisms that share common characteristics)
programmed cell death
Describes a process where cells destroy themselves when they are no longer needed.