detection-and-isolation-of-apoptotic-bodies-to-high-purity

Detection and Isolation of Apoptotic Bodies to High Purity

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

La Trobe University

Disassembly of apoptotic cells into smaller fragments (a form of extracellular vesicle called apoptotic bodies) can facilitate removal of apoptotic debris and intercellular communication. However, the mechanism underpinning this process is unclear. While observing monocytes undergoing apoptosis by time-lapse microscopy, we discovered a new type of membrane protrusion that resembles a 'beads-on-a-string' structure. Strikingly, the 'beads' are frequently sheared off the 'string' to form apoptotic bodies. Generation of apoptotic bodies via this mechanism can facilitate a sorting process and results in the exclusion of nuclear contents from apoptotic bodies. Mechanistically, generation of 'beads-on-a-string' protrusion is controlled by the level of actomyosin contraction and apoptopodia formation. Furthermore, in an unbiased drug screen, we identified the ability of sertraline (an antidepressant) to block the formation of 'beads-on-a-string' protrusions and apoptotic bodies. These data uncover a new mechanism of apoptotic body formation in monocytes and also compounds that can modulate this process. 

A novel mechanism of generating extracellular vesicles during apoptosis via a beads-on-a-string membrane structure.

The use of annexin A5 (A5) and either propidium iodide or 7-aminoactinomycin D (PI/7-AAD) stains to measure cell death by flow cytometry has been considered the gold standard by most investigators. However, this widely used method often makes the assumption that there are only three types of particles in a sample: viable, apoptotic and necrotic cells. To study the progression of cell death in greater detail, in particular how apoptotic cells undergo fragmentation to generate membrane-bound vesicles known as apoptotic bodies, we established a flow cytometry-based protocol to accurately and rapidly measure the cell death process. This protocol uses a combination of A5 and TO-PRO-3 (a commercially available nucleic acid-binding dye that stains early apoptotic and necrotic cells differentially), and a logical seven-stage analytical approach to distinguish six types of particles in a sample, including apoptotic bodies and cells at three different stages of cell death. The protocol requires 1-5 h for sample preparation (including induction of cell death), 20 min for staining and 5 min for data analysis. 

Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry.

The disassembly of an apoptotic cell into subcellular fragments, termed apoptotic bodies (ApoBDs), is a hallmark of apoptosis. Although the generation of ApoBDs is generally understood as being stochastic, it is becoming increasingly clear that ApoBD formation is a highly regulated process involving distinct morphological steps and molecular factors. Functionally, ApoBDs could facilitate the efficient clearance of apoptotic material by surrounding phagocytes as well as mediate the transfer of biomolecules including microRNAs and proteins between cells to aid in intercellular communications. Therefore, the formation of ApoBDs is an important process downstream from apoptotic cell death. We discuss here the mechanisms and functions of apoptotic cell disassembly.

Disassembly of the Dying: Mechanisms and Functions.

Apoptotic bodies (ApoBDs) are membrane-bound extracellular vesicles that can mediate intercellular communication in physiological and pathological settings. By combining recently developed analytical strategies with fluorescence-activated cell sorting (FACS), we have developed a method that enables the isolation of ApoBDs from cultured cells to 99% purity. In addition, this approach also enables the identification and isolation of cell type-specific ApoBDs from tissue, bodily fluid and blood-derived samples.

Isolation of cell type-specific apoptotic bodies by fluorescence-activated cell sorting.

Defining the morphologic features and products of cell disassembly during apoptosis.

Over 200 billion cells undergo apoptosis every day in the human body in order to maintain tissue homeostasis. Increased apoptosis can also occur under pathological conditions including infection and autoimmune disease. During apoptosis, cells can fragment into subcellular membrane-bound vesicles known as apoptotic bodies (ApoBDs). We recently developed a flow cytometry-based method to accurately differentiate ApoBDs from other particles (e.g. cells and debris). In the present study, we aim to further characterize subsets of ApoBDs based on intracellular contents and cell type-specific surface markers. Utilizing a flow cytometry-based approach, we demonstrated that intracellular contents including nuclear materials and mitochondria are distributed to some, but not all ApoBDs. Interestingly, the mechanism of ApoBD formation could affect the distribution of intracellular contents into ApoBDs. Furthermore, we also showed that ApoBDs share the same surface markers as their cell of origin, which can be used to distinguish cell type-specific ApoBDs from a mixed culture. These studies demonstrate that ApoBDs are not homogeneous and can be divided into specific subclasses based on intracellular contents and cell surface markers. The described flow cytometry-based method to study ApoBDs could be used in future studies to better understand the function of ApoBDs.

Georgia K Atkin-Smith

Department of Biochemistry and Genetics,
La Trobe Institute for Molecular Science,
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science

La Trobe University

Detection and Isolation of Apoptotic Bodies to High Purity

Georgia K Atkin-Smith

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry and Genetics,La Trobe Institute for Molecular Science,Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science

La Trobe University

Detection and Isolation of Apoptotic Bodies to High Purity

Department of Biochemistry and Genetics,
La Trobe Institute for Molecular Science,
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science