Please note that this presentation aims at summarizing currently available data only without the claim of representing established knowledge. Thus information presented in this course may be subject to change in the future due to the rapid evolvement of the field of extracellular vesicles. This lecture will focus on the release of extracellular vesicles or EVS during apoptosis, a form of programmed cell death, and cell senescence where normal cells cease to divide, in particular their roles in physiological processes. Section 1 will be presented by Dr. Ivan Poon on the mechanism and function of apoptotic cell disassembly and section 2 will be presented by Professor Hidetoshi Tahara on EVs from senescence cells. My name is KJarolina Sigmagy. I am an adjunct board member for ISEF and a member of the educational team. I will narrate this lecture. Let's begin. Doctor Ivan Poon is laboratory head from the La Trobe Institute for Molecular Science Australia and his lecture focuses on how and why apoptotic cells need to break apart into smaller fragments known as apoptotic bodies. Apoptosis is a form of programmed cell death and billions of cells will undergo apoptosis daily as part of the physiological homeostasis to eliminate unwanted cells. Apoptosis can occur in essentially all tissues as part of normal development. Apoptosis can also occur in a number of disease settings including infection, cancer, acero sclerosis, and autoimmunity. The focus of this section of the lecture is what happens at later stages of apoptosis where certain cell types can undergo fragmentation to generate sub cellular membrane bound extracellular fascicles known as apoptotic bodies or ApoBD. The process of generating apoptotic bodies is called apoptotic cell disassembly. It should be noted that apoptotic bodies are generally described as 1 to 5 microns in diameter and expose the phospholipid on its surface. The mechanism of apoptotic body formation and the function of this process will be discussed in detail in the next few slides. The fragmentation of an apoptotic cell into apoptotic bodies had been thought for many years to be a stochastic process, a process driven predominantly by apoptotic membrane blebbing. Phospho membrane blebbing is the morphological hallmark of apoptosis and it is a cellular process that describes the formation and retraction of plasma membrane bulges at the cell periphery. Although membrane blebbing is a key step prior to the formation of apoptotic bodies, it is becoming more recognized recently that addition processes are required to generate apoptotic bodies. Following membrane blebbing, the formation of thin apoptotic membrane protrusions has also been described recently to play a key role in driving and regulating apoptotic body formation. Apoptotic membrane protrusion can appear in a number of morphological forms including rigid protrusions called microtubule spikes, string like protrusions called apoptopodia., and beads on a string protrusions called beaded apoptopodia. Formation of this apoptotic membrane protrusion is necessary to separate blebs or generate a string of vesicles. Lastly, dissociation of blebs or vesicles from these apoptotic membrane protrusions to generate distinct apoptotic bodies marks the final cell fragmentation stage of apoptotic cells. Thus the apoptotic cell disassembly process can be divided into three sequential steps governed by distinct morphological changes. Step 1 apoptotic membrane blebbing, step 2 apoptotic membrane protrusion formation, step 3 cell fragmentation or apoptotic body formation. In the next few slides, molecular processes that regulates these steps of apoptotic cell disassembly will be discussed further. Apoptotic membrane blebbing is regulated by a number of processes including hydrostatic pressure, which facilitates the movement of intracellular fluids into membrane blebs and promotes bleb inflation. Actomyosin contraction, a process that describes the generation of contractile force through the interaction between F-actin and myosin is also important for the formation of membrane blebs during apoptosis. It should be noted that actomyosin contraction during apoptosis is regulated by a number of kinases including rho-associated protein kinase 1, ROCK1 ,myosin light-chain kinase MLCK, limb domain kinase 1, LIMK1 and p21 activated kinase, PAK2. Lastly, assembly of microtubules also regulate cytoskeletal dynamics during apoptosis to control the cyclic extension of blebs at the cell surface. Depending on the cell type, different type of apoptotic membrane protrusion could be formed t o facilitate apoptotic body formation. Generation of microtubule spikes can be observed on apoptotic epithelial cells. As the name suggested, assembly of microtubule during apoptosis is key for microtubule spike formation. Formation of apoptopodia can be observed on apoptotic cells, and fibroblasts where beaded apoptopodia can be found on apoptotic monocytes. Both apoptopodia and beaded apoptopodia are negatively regulated by the activity of pannexin 1 membrane channels, PANX1, and positively regulated by vesicular trafficking. The final stage of apoptotic cell disassembly is the dissociation of apoptotic bodies from the cell body or neighboring apoptotic bodies. Although the precise mechanism controlling this step is not well-defined, it may involve both cell-extrinsic and-cell intrinsic factors, such as shear force, abscission-like process, as well as interaction with neighboring phagocytes. Collectively, apoptotic body formation is a highly coordinated process controlled by a merit of mechanisms. Functionally, the formation of apoptotic bodies has been proposed to mediate two key processes. One, to promote efficient removal of apoptotic cells. Two, to carry biomolecules such as nucleic acids and proteins to facilitate intercellular communication. Under normal physiological conditions, apoptotic cells are rapidly removed by phagocytes, a process known as cell clearance. If apoptotic cells are not cleared in a timely manner, the dying cells can undergo secondary necrosis, where the plasma membrane becomes permeabilized, leading to the release of pro-inflammatory intracellular contents. Thus, intercell clearance has been linked to a number of chronic inflammatory diseases, including systemic lupus, erythematosus, atherosclerosis, and colitis. To aid the cell clearance process, apoptotic cells can release find me signals, such as nucleotides to recruit nearby phagocytes toward the site of cell death. Following phagocyte recruitment, engulfment of apoptotic cells is triggered through the recognition of eat me signals, in particular phosphatidylserine, exposed on the surface of apoptotic cells. In addition of this molecular mechanism of cell clearance, apoptotic cells disassembly has also been proposed to aid apoptotic cell removal, possibly by generating bite-sized pieces. For example, the apoptotic bodies that are more readily engulfed by phagocytes. It has been well described in the literature that other types of EVs such as exosomes and microvesicles can mediate intercellular communication by trafficking cellular contents between cells. Likewise, apoptotic bodies can also function in a similar manner. For example, oncogenes from apoptotic lymphoma cells can be packaged into apoptotic bodies. The transfer of oncogenes via apoptotic bodies to recipient cells can subsequently promote tumor formation in vivo. In addition to DNA, certain microRNA can also be enriched in apoptotic bodies generated from apoptotic endothelial cells. And their transfer to recipient endothelial cells can promote tissue repair. Apoptotic bodies can also have a protein, such as cytokine, to regulate inflammation. In summary, apoptotic disassembly is a key process downstream of apoptosis. ApoBD formation is a highly complex process regulated by a number of well-coordinated morphological steps, including apoptotic membrane blebbing, apoptotic protrusion formation, and fragmentation. The formation of ApoBD can aid cell clearance and intercellular communication. The second lecture will focus on the cell senescence, when normal cells cease to divide. This section will be presented by Professor Hidetoshi Tahara on EVs from senescent cells. Most somatic cell have limited lifespan in vitro and in vivo. Cell senescence is the loss of ability to divide after a finite number of divisions. During cellular senescence, normal cells, such as human diploid fibroblasts, change their morphology from a spindle shape to flatten and an enlarged morphology, accompanied with growth suppression and SASP factor production. Multiple signals are involved in the induction of cellular senescence. It is well known that H-rasV12 is an oncogene in cancer cells. Interestingly, introduction of H-rasV12 in human normal cells induce cellular senescence, so-called oncogene-induced senescence. Our body is under constant oxidative attack from reactive oxygen species or RAS. Therefore, understanding of oxidative stress induced senescence is important for in vivo aging. Telomere reduction is well known in most somatic cells, and induce cellular senescence by activating cell cycle inhibitors such as p16 and p21. Multiple senescence inducers induce cellular senescence accompanied with telomere reduction. Major morphological change is enlargement of cells. Extracellular vesicle production is increased during cellular senescence, together with an increase of the production of SASP factors. The EV profile is also altered during cellular senescence. Senescence like cells can be found in premalignant tumor, but these cells disappear when tumor cells become malignant. EVs and SASP factors are known to be involved in tumor microenvironments. Senescent cells may play important roles for tumor progression in tumor microenvironments. It is well known that extracellular vesicles contain various lengths of genomic DNA fragments. During cellular senescence, extracellular vesicle-mediated DNA secretion increases. Telomere repair DNA, TTAGGG, can be transcribed as telomeric repeats containing RNA or TERRA, in response to developmental changes and cellular stress conditions. TERRA has been implicated in telomere length regulation and DNA damage signaling. TERRA can also be found in extracellular fractions, and stimulate innate immune signaling. Other functions, such as cancer cell proliferation, calcification, and myelination reduction, are also reported. In summary, senescent cells have a limited life span both in vitro and in vivo. SV40/E6/E7 elongate the life-span, but still reduce telomere length. Therefore, these cells have a limited lifespan in human cells. Telomerase activation by TERT induce cell immortalization, accompanied with telomere elongation. Multiple factors such as oncogene RAS, oxidative stress, telomere dysfunction, and DNA damage signals are involved in the induction of senescence. Increased EV secretion was observed during senescence. EVs derived from senescent cells involved in tumor progressions, in tumor microenvironments. Senescent cell derived EVs have multiple functions, such as DNA damage activation, immune cell activation. Thank you for listening to this lecture.
