in-vitro-cleavage-assays-using-purified-recombinant-drosophila

<em>In Vitro</em> Cleavage Assays using Purified Recombinant <em>Drosophila</em> Caspases for Substrate Screening

Caspases are very specific cell death proteases that are involved in apoptotic and non-apoptotic processes. While the role of caspases during apoptosis ...

UMASS Chan Medical School

Although apoptosis is mechanistically well understood, a comprehensive understanding of how cells modulate their susceptibility toward apoptosis in a developing tissue is lacking. Here, we reveal striking dynamics in the apoptotic susceptibilities of different cell types in the Drosophila retina over a period of only 24 hr. Mitotic cells are extremely susceptible to apoptotic signals, while postmitotic cells have developed several strategies to promote survival. For example, photoreceptor neurons accumulate the inhibitor of apoptosis, Diap1. In unspecified cells, Cullin-3-mediated degradation keeps Diap1 levels low. These cells depend on EGFR signaling for survival. As development proceeds, developmentally older photoreceptors degrade Diap1, resulting in increased apoptosis susceptibility. Finally, R8 photoreceptors have very efficient survival mechanisms independent of EGFR or Diap1. These examples illustrate how complex cellular susceptibility toward apoptosis is regulated in a developing organ. Similar complexities may regulate apoptosis susceptibilities in mammalian development, and tumor cells may take advantage of it. 

Multiple mechanisms modulate distinct cellular susceptibilities toward apoptosis in the developing Drosophila eye.

Apoptosis is a carefully choreographed process of cellular self-destruction in the absence of inflammation. During the death process, apoptotic cells actively communicate with their environment, signaling to both their immediate neighbors as well as distant sentinels. Some of these signals direct the anti-inflammatory immune response, instructing specific subsets of phagocytes to participate in the limited and careful clearance of dying cellular debris. These immunomodulatory signals can also regulate the activation state of the engulfing phagocytes. Other signals derived from apoptotic cells contribute to tissue growth control with the common goal of maintaining tissue integrity. Derangements in these growth control signals during prolonged apoptosis can lead to excessive cell loss or proliferation. Here, we highlight some of the most intriguing signals produced by apoptotic cells during the course of normal development as well as during physiological disturbances such as atherosclerosis and cancer. 

The Sound of Silence: Signaling by Apoptotic Cells.

Apoptosis-induced proliferation (AiP) is a compensatory mechanism to maintain tissue size and morphology following unexpected cell loss during normal development, and may also be a contributing factor to cancer and drug resistance. In apoptotic cells, caspase-initiated signaling cascades lead to the downstream production of mitogenic factors and the proliferation of neighboring surviving cells. In epithelial cells of Drosophila imaginal discs, the Caspase-9 ortholog Dronc drives AiP via activation of Jun N-terminal kinase (JNK); however, the specific mechanisms of JNK activation remain unknown. Here we show that caspase-induced activation of JNK during AiP depends on an inflammatory response. This is mediated by extracellular reactive oxygen species (ROSs) generated by the NADPH oxidase Duox in epithelial disc cells. Extracellular ROSs activate Drosophila macrophages (hemocytes), which in turn trigger JNK activity in epithelial cells by signaling through the tumor necrosis factor (TNF) ortholog Eiger. We propose that in an immortalized ("undead") model of AiP, signaling back and forth between epithelial disc cells and hemocytes by extracellular ROSs and TNF/Eiger drives overgrowth of the disc epithelium. These data illustrate a bidirectional cell-cell communication pathway with implication for tissue repair, regeneration, and cancer.

Extracellular Reactive Oxygen Species Drive Apoptosis-Induced Proliferation via Drosophila Macrophages.

ATG1 belongs to the Uncoordinated-51-like kinase protein family. Members of this family are best characterized for roles in macroautophagy and neuronal development. Apoptosis-induced proliferation (AiP) is a caspase-directed and JNK-dependent process which is involved in tissue repair and regeneration after massive stress-induced apoptotic cell loss. Under certain conditions, AiP can cause tissue overgrowth with implications for cancer.

Autophagy-independent function of Atg1 for apoptosis-induced compensatory proliferation.

The myotonic dystrophies are prototypic toxic RNA gain-of-function diseases. Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are caused by different unstable, noncoding microsatellite repeat expansions - (CTG) in  and (CCTG) in  Although transcription of mutant repeats into (CUG) or (CCUG) appears to be necessary and sufficient to cause disease, their pathomechanisms remain incompletely understood. To study the mechanisms of (CCUG) toxicity and develop a convenient model for drug screening, we generated a transgenic DM2 model in the fruit fly  with (CCUG) repeats of variable length (=16 and 106). Expression of noncoding (CCUG), but not (CCUG), in muscle and retinal cells led to the formation of ribonuclear foci and mis-splicing of genes implicated in DM pathology. Mis-splicing could be rescued by co-expression of human MBNL1, but not by CUGBP1 (CELF1) complementation. Flies with (CCUG) displayed strong disruption of external eye morphology and of the underlying retina. Furthermore, expression of (CCUG) in developing retinae caused a strong apoptotic response. Inhibition of apoptosis rescued the retinal disruption in (CCUG) flies. Finally, we tested two chemical compounds that have shown therapeutic potential in DM1 models. Whereas treatment of (CCUG) flies with pentamidine had no effect, treatment with a PKR inhibitor blocked both the formation of RNA foci and apoptosis in retinae of (CCUG) flies. Our data indicate that expression of expanded (CCUG) repeats is toxic, causing inappropriate cell death in affected fly eyes. Our  DM2 model might provide a convenient tool for  drug screening.

(CCUG) RNA toxicity in a  model of myotonic dystrophy type 2 (DM2) activates apoptosis.

Apoptosis-induced compensatory proliferation (AiP) is a form of compensatory proliferation that is triggered by apoptotic cell death to maintain tissue homeostasis. As such, AiP is essential for many tissue repair processes including regeneration. The apoptotic effectors, termed caspases, not only execute apoptosis, but are also directly involved in the generation of the signals required for AiP. Reactive oxygen species (ROS) play an important role for regenerative processes. Recently, it was shown in Drosophila that apoptotic caspases can mediate the generation of ROS for promoting AiP. This review summarizes and discusses these findings in the context of regenerative processes and cancer.

An unexpected friend - ROS in apoptosis-induced compensatory proliferation: Implications for regeneration and cancer.

Caspases are best characterized for their function in apoptosis. However, they also have non-apoptotic functions such as apoptosis-induced proliferation (AiP), where caspases release mitogens for compensatory proliferation independently of their apoptotic role. Here, we report that the unconventional myosin, Myo1D, which is known for its involvement in left/right development, is an important mediator of AiP in Drosophila. Mechanistically, Myo1D translocates the initiator caspase Dronc to the basal side of the plasma membrane of epithelial cells where Dronc promotes the activation of the NADPH-oxidase Duox for reactive oxygen species generation and AiP in a non-apoptotic manner. We propose that the basal side of the plasma membrane constitutes a non-apoptotic compartment for caspases. Finally, Myo1D promotes tumor growth and invasiveness of the neoplastic scrib Ras model. Together, we identified a new function of Myo1D for AiP and tumorigenesis, and reveal a mechanism by which cells sequester apoptotic caspases in a non-apoptotic compartment at the plasma membrane.

Plasma Membrane Localization of Apoptotic Caspases for Non-apoptotic Functions.

Apoptosis has long been regarded as a tumor suppressor mechanism and evasion from apoptosis is considered to be one hallmark of cancer. However, this principle is not always consistent with clinical data which often illustrate a correlation between apoptosis and poor prognosis. Work in the last 15 years has provided an explanation for this apparent paradox. Apoptotic cells communicate with their environment and can produce signals which promote compensatory proliferation of surviving cells. This behavior of apoptotic cells is important for tissue regeneration in several model organisms, ranging from hydra to mammals. However, it may also play an important feature for tumorigenesis and tumor relapse. Several distinct forms of apoptosis-induced compensatory proliferation (AiP) have been identified, many of which involve reactive oxygen species (ROS) and immune cells. One type of AiP, "undead" AiP, in which apoptotic cells are kept in an immortalized state and continuously divide, may have particular relevance for tumorigenesis. Furthermore, given that chemo- and radiotherapy often aim to kill tumor cells, an improved understanding of the effects of apoptotic cells on the tumor and the tumor environment is of critical importance for the well-being of the patient. In this review, we summarize the current knowledge of AiP and focus our attention on recent findings obtained in Drosophila and other model organisms, and relate them to tumorigenesis.

Two Sides of the Same Coin - Compensatory Proliferation in Regeneration and Cancer.

Macrophages are a major immune cell type infiltrating tumors and promoting tumor growth and metastasis. To elucidate the mechanism of macrophage recruitment, we utilize an overgrowth tumor model ("undead" model) in larval Drosophila imaginal discs that are attached by numerous macrophages. Here we report that changes to the microenvironment of the overgrown tissue are important for recruiting macrophages. First, we describe a correlation between generation of reactive oxygen species (ROS) and damage of the basement membrane (BM) in all neoplastic, but not hyperplastic, models examined. ROS and the stress kinase JNK mediate the accumulation of matrix metalloproteinase 2 (Mmp2), damaging the BM, which recruits macrophages to the tissue. We propose a model where macrophage recruitment to and activation at overgrowing tissue is a multi-step process requiring ROS- and JNK-mediated Mmp2 upregulation and BM damage. These findings have implications for understanding the role of the tumor microenvironment for macrophage activation.

Basement membrane damage by ROS- and JNK-mediated Mmp2 activation drives macrophage recruitment to overgrown tissue.

We reveal surprising similarities between homeostatic cell turnover in adult Drosophila midguts and "undead" apoptosis-induced compensatory proliferation (AiP) in imaginal discs. During undead AiP, immortalized cells signal for AiP, allowing its analysis. Critical for undead AiP is the Myo1D-dependent localization of the initiator caspase Dronc to the plasma membrane. Here, we show that Myo1D functions in mature enterocytes (ECs) to control mitotic activity of intestinal stem cells (ISCs). In Myo1D mutant midguts, many signaling events involved in AiP (ROS generation, hemocyte recruitment, and JNK signaling) are affected. Importantly, similar to AiP, Myo1D is required for membrane localization of Dronc in ECs. We propose that ECs destined to die transiently enter an undead-like state through Myo1D-dependent membrane localization of Dronc, which enables them to generate signals for ISC activity and their replacement. The concept of transiently "undead" cells may be relevant for other stem cell models in flies and mammals.

Transiently "Undead" Enterocytes Mediate Homeostatic Tissue Turnover in the Adult Drosophila Midgut.

The initiator caspase Dronc is the only CARD-domain containing caspase in Drosophila and is essential for apoptosis. Here, we report that homozygous dronc mutant adult animals are short-lived due to the presence of a poorly developed, defective and leaky intestine. Interestingly, this mutant phenotype can be significantly rescued by enteroblast-specific expression of dronc in dronc mutant animals, suggesting that proper Dronc function specifically in enteroblasts, one of four cell types in the intestine, is critical for normal development of the intestine. Furthermore, enteroblast-specific knockdown of dronc in adult intestines triggers hyperplasia and differentiation defects. These enteroblast-specific functions of Dronc do not require the apoptotic pathway and thus occur in a non-apoptotic manner. In summary, we demonstrate that an apoptotic initiator caspase has a very critical non-apoptotic function for normal development and for the control of the cell lineage in the adult midgut and therefore for proper physiology and homeostasis.

Non-apoptotic enteroblast-specific role of the initiator caspase Dronc for development and homeostasis of the Drosophila intestine.

Although there are over a dozen types of cell death known, there is clearly more to discover in this field. In this issue of PLOS Biology, erebosis is identified as a new type of cell death involved in tissue homeostasis of the adult Drosophila intestine.

Erebosis is a new type of cell death for tissue homeostasis in the Drosophila intestine.

Drosophila Toll-1 and all mammalian Toll-like receptors regulate innate immunity. However, the functions of the remaining eight Toll-related proteins in Drosophila are not fully understood. Here, we show that Drosophila Toll-9 is necessary and sufficient for a special form of compensatory proliferation after apoptotic cell loss (undead apoptosis-induced proliferation [AiP]). Mechanistically, for AiP, Toll-9 interacts with Toll-1 to activate the intracellular Toll-1 pathway for nuclear translocation of the NF-κB-like transcription factor Dorsal, which induces expression of the pro-apoptotic genes reaper and hid. This activity contributes to the feedback amplification loop that operates in undead cells. Given that Toll-9 also functions in loser cells during cell competition, we define a general role of Toll-9 in cellular stress situations leading to the expression of pro-apoptotic genes that trigger apoptosis and apoptosis-induced processes such as AiP. This work identifies conceptual similarities between cell competition and AiP.

Article	Year
Multiple mechanisms modulate distinct cellular susceptibilities toward apoptosis in the developing Drosophila eye. Developmental cell\| PubMed ID: 24981611	2014
The Sound of Silence: Signaling by Apoptotic Cells. Current topics in developmental biology\| PubMed ID: 26431570	2015
Extracellular Reactive Oxygen Species Drive Apoptosis-Induced Proliferation via Drosophila Macrophages. Current biology : CB\| PubMed ID: 26898463	2016
Autophagy-independent function of Atg1 for apoptosis-induced compensatory proliferation. BMC biology\| PubMed ID: 27542914	2016
(CCUG) RNA toxicity in a model of myotonic dystrophy type 2 (DM2) activates apoptosis. Disease models & mechanisms\| PubMed ID: 28623239	2017
An unexpected friend - ROS in apoptosis-induced compensatory proliferation: Implications for regeneration and cancer. Seminars in cell & developmental biology\| PubMed ID: 28688927	2018
Plasma Membrane Localization of Apoptotic Caspases for Non-apoptotic Functions. Developmental cell\| PubMed ID: 29787709	2018
Two Sides of the Same Coin - Compensatory Proliferation in Regeneration and Cancer. Advances in experimental medicine and biology\| PubMed ID: 31520349	2019
Basement membrane damage by ROS- and JNK-mediated Mmp2 activation drives macrophage recruitment to overgrown tissue. Nature communications\| PubMed ID: 32686670	2020
Transiently "Undead" Enterocytes Mediate Homeostatic Tissue Turnover in the Adult Drosophila Midgut. Cell reports\| PubMed ID: 33238125	2020
Non-apoptotic enteroblast-specific role of the initiator caspase Dronc for development and homeostasis of the Drosophila intestine. Scientific reports\| PubMed ID: 33514791	2021
Erebosis is a new type of cell death for tissue homeostasis in the Drosophila intestine. PLoS biology\| PubMed ID: 35472045	2022
Toll-9 interacts with Toll-1 to mediate a feedback loop during apoptosis-induced proliferation in Drosophila. Cell reports\| PubMed ID: 35584678	2022

Andreas Bergmann

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