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Autophagic and Apoptotic Features During Programmed Cell Death in the Fat Body of the Tobacco Hornworm (Manduca Sexta)

Posted on: Thursday, 3 June 2004, 06:00 CDT

Programmed cell death; Manduca sexta; autophagy; apoptosis; DNA fragmentation

Two major pathways of programmed cell death (PCD) - the apoptotic and the autophagic cell death - were investigated in the decomposition process of the larval fat body during the 5th larval stage of Manduca sexta. Several basic aspects of apoptotic and autophagic cell death were analyzed by morphological and biochemical methods in order to disclose whether these mechanisms do have shared common regulatory steps. Morphological examination revealed the definite autophagic wave started on day 4 followed by DNA fragmentation as demonstrated by agarose gel electrophoresis and TUNEL assay. By the end of the wandering period the cells were filled with autophagic vacuoles and protein granules of heterophagic origin and the vast majority of the nuclei were TUNEL-positive. No evidence was found of other aspects of apoptosis, e.g. activation of executioner caspases. Close correlation was disclosed between the onset of autophagy and the nuclear accumulation of the ubiquitin- proteasome system.

Introduction

The term "programmed cell death" (PCD) was introduced in a series of papers by Lockshin and Williams (1965a, b) as the activation of an intrinsic genetic program leading to the temporally regulated degeneration of the intersegmental muscles in developing insects. The general characterization of PCD can be summarized as follows. It can be described as the high-level coordination of events leading to the temporally and spatially well-determined elimination of certain cells, tissues or organs. This energy-dependent process requires de novo gene expression in the cells prior to the execution of the "suicide", consequently it can he blocked by inhibitors of transcription or translation (Tata, 1966; Pratt and Greene, 1976; Colbert and Young, 1986; Oppenheim et al., 1990). It is a strictly regulated procedure, which is controlled by the withdrawal of trophic factors, or by the production of factors/hormones activating the cell death genes in the target cells (Lockshin. 1969; Truman ct al., 1992).

Two major mechanisms of PCD have been well classified: the apoptotic cell death (type 1) and the autophagic cell death (type 2) (Beaulaton and Lockshin, 1982: Schwartz et al., 1993). The term apoptosis was originally introduced by Kerr et al. (1972) to describe the morphological events leading to the cell death in pathological situations. Later on the terms apoptosis and PCD tended to become synonymous in the literature although they are definitely not equivalent (Schwartz et al., 1993; Lockshin and Zakeri, 1994).

The distinguishing features of apoptosis are appearance of cytoplasmic blebs on the surface of dying cells and the formation of apoptotic bodies which are phagocytosed subsequently by the surrounding cells (Kerr et al., 1972) and the characteristic fragmentation of DNA due to the internucleosomal cleavage by activated endonucleases (Cohen and Duke, 1984).

The molecular research on apoptosis in mammals and nematodes had revealed a large number of proteins and genes, e.g. members of ICE/ ced-3 and Bcl-2/ced-9 gene families, which have been implicated as essential components in the control of this active cell death.

The ICE/ced-3 gene family comprises the interleukin 1[beta]- converting enzyme (ICE)-like cysteine proteases or caspases; and apoptosis is associated with the hierarchical activation of these enzymes. Phylogenetic analysis revealed three subfamilies of caspases. 1) The ICE subfamily involved in cytokine activation (Cerretti et al., 1992). 2) The ced-3/CPP32 subfamily comprises two groups based on their position within the proteolytic cascade. The downstream, executioner caspases, with short prodomain, are involved in promoting the apoptotic phenotype, e.g. the strong DNA fragmentation (Fernandes-Alnemri et al., 1994). The upstream, initiator caspases, with long prodomain, amplify the death signals and activate the executioner caspases by limited proteolysis (Li et al., 1997). 3) The third subfamily - ICH-1/Nedd2, with only one member (caspase-2) - has considerable structural similarity to some ced3/CPP32 members and the pro-caspase-2 can be processed by other caspase family members (Harvey et al., 1996).

Evidence for a role of Bcl-2/ced-9 family proteins in cell survival was presented in mammals (Ngan et al., 1988). The Bel2/ced- 9 family of proteins are substantial components of the mitochondrial regulation pathway of apoptosis. The apoptotic caspase cascade is regulated by these proteins via the adaptor protein APAF-1 (apoptotic protease-activating factor 1) (Zou et al., 1997).

Another mechanism of PCD is the ubiquitin-dependent proteolysis observed in the intersegmental muscles of the tobacco hornworm (Manduca sexta) following the adult eclosion in response to the decrease in the 20-hydroxyecdysone titer. In the muscle cells committed to die an increase in the expression of polyubiquitin was described which has been shown as an essential mediator of proteolysis (Myer and Schwartz, 1996). Ubiquitin was originally discovered as a molecular tag that could be covalently bound to certain target proteins to mark them for degradation by the proteasome. The expression of the multicatalytic proteinases responsible for the uhiquitin-dependent proteolysis of intersegmental muscles was also studied and an eightfold increase was noted. This rise was triggered by the same hormonal signals that mediate ubiquitin expression and cell death (Jones et al., 1995). Increased polyubiquitin expression under the effect of thyroxine has been shown to be involved in tissue regression in the tail tips of Rana catesbeiana tadpoles (Phillips and Platt, 1994).

In contrast to apoptosis, the genetic and molecular background of the type 2 (autophagic) mechanism of PCD is not well known, although, the morphological and biochemical features of autophagocytosis are characterized in detail. Autophagy is defined as the process by which n portion of the cytosol or certain organelles are sequestered in a random manner by socalled isolatory membranes. The autophagosomes thus formed, subsequently fuse with lysosomes forming autophagic vacuoles, inside which the macromolecules of the engulfed organelles are digested at acidic pH by the lysosomal enzymes. Primarily, autophagy is regarded as a cellular response to starvation, which promotes the survival of the cell (Bowen et al., 1976; Pfeifer, 1976). Later on it has been discovered that in the cells undergoing PCD in developing systems autophagic vacuoles appear degrading practically all of the scytoplasmic organelles.

In our laboratory the mechanisms of PCD is studied in the larval organs of metamorphosing Lepidopteran insects. It has been shown that the cells of the larval fat body decompose via autophagocytosis in a temporally and spatially well-determined manner. This process is stimulated by the molting hormone dunder in vivo and in vitro conditions, and is inhibited by juvenile hormone (Sass and Kovacs, 1975, 1977). The physiological process and the hormone-stimulated autophagocytosis can be blocked by inhibition of transcription and translation (Sass and Kovacs, 1980). Thus, autophagic degradation of larval organs during metamorphosis can be regarded as a form of programmed cell death, as it meets all the criteria of PCD mentioned above.

Some evidence suggests that although the salivary gland, the intersegmental muscles and the prothoracic glands decompose by autophagy during metamorphosis, the fragmentation of genomic DNA can also be observed in these cell types (Zakeri et al., 1993; Dai and Gilbert, 1997, 1999).

On the basis of these considerations we investigated in this study the DNA fragmentation, the activation of the above mentioned executioner caspases, and the ubiquitin-proteasome system before the onset and during the autophagic degradation of the larval fat body cells in a holometabolous Lepidopteran insect, Manduca sexta to disclose whether these different mechanisms of PCD do or do not have shared regulatory steps in this system.

Materials and methods

Experimental animals

Tobacco hornworm (Manduca sexta) eggs were kindly provided by Prof. S. E. Reynolds (University of Bath, UK). Larvae were reared according Io his instructions, based on the techniques described by Bell and Joachim (1976), on a wheat germ-based artificial diet at 25C, 17L:7D photoperiod and 60% relative humidity. Animals were selected that cedysed to the 5th larval stage. Growth was completed on day 4 subsequent to which larvae initiated wandering behavior. Different stages of development were recognized by a staging scheme adapted from (Samuels et al., 1993). The animals were sampled from each physiological period of the 5th larval stage. The feeding period was represented by the animals collected on days 0, 1, 2, 3 and 4 (V0, V1, V2, V3, V4). The transition state - the boundary of the feeding and the wandering period -was bound to day 5 (V5/W0). The early wandering period was represented by animals collected on days 6 (V6/W1) and 7 (V7/W2) - and the late wandering period by animals collected on days 8 (V8/W3) and 9 (V9/W4). Occasionally, samples were used from the 4th larval stage and early pupal stage for comparison.

Sample preparation and light microscopic morphological analysis

The \material for histological investigations was prepared as follows. The animals were fixed in Bouin fixative for 6 hours at room temperature. Fixatives were injected into the body cavity, and the animals were subsequently immersed into a large volume of Bouin fixative. After 2 hours the body was cut into three pieces and fixed further. Fixed materials were embedded in Paraplast (Dulbecco). Five- m serial sections were cut, placed on poly-L-lysine-coated slides and dried overnight at 39C. After deparaffinating by xylene and alcohol, the samples were rehydrated and stained with haematoxylin- eosin.

Electron microscopic analysis

Fat body lobes were dissected from chilled Manduca sexla larvae in the feeding period (day 2 of the 5th larval stage) under a stereomicroscope. Tissue samples were fixed in 4% paraformaldehyde, 0.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 1 h at 4C. After rinsing with 0.1 M cacodylate buffer, the fat body pieces were postfixed for 1 h in 0.5% osmium tetroxide. After dehydration in a graded series of ethanol and then in propylene dioxide at room temperature, the tissue was embedded in Durcupan (ACM, Fluka, Buchs, Switzerland). Fifty-nanometer sections were cut. Grids were stained with uranyl acetate for 8 min, lead citrate for 2 min and then blotted dry. Preparations were analyzed with a JEOL electron microscope (JEOL 100CX II Electron Microscope) at an acceleration voltage of 60 kV.

Detection of DNA fragmentation

Apoptotic cells were identified in situ using the TUNEL (TdT- mediated dUTP-biotin Nick End Labeling) technique. Paraffin- embedded samples were prepared in the same manner as it was described above. The applied TUNEL staining was based on the protocol described by Migheli et al. (1997). The rehydrated sections were rinsed in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) twice for 10 minutes at room temperature (RT). Protemase-K (2 mg/ml) digestion was applied as a pretreatment for 20 minutes at RT. The sections were rinsed in TE subsequently. A special, two-component TdT buffer was used for the appropriate preparation of the sections prior to the TdT (terminal deoxynucleotidyl transferase) reaction. Fresh TdT buffer was prepared by mixing equal volumes of stock solutions 1 and 2 (Solution 1 (2 ) 50 mM Tris-HCl, 0.4 M potassium cacodylate, pH 6.6; Solution 2 (2 ) 5 mM CoCl2). The TUNEL assay was carried out using the In situ Cell Death Detection Kit, AP (Roche Diagnostics GmbH, Mannheim, Germany). The kit was used in accordance with the instructions provided by the manufacturer. Sections were incubated for 2 hours at 37 C, subsequently rinsed twice in 2% SSC (15 min) and once in TBS (15 min), and blocked with 2% BSA (20 min). The signal generated by fluorescein-labeled dUTP incorporated at the nicked ends of fragmented DNA during the TdT reaction was not easily detectable on the background of high fixative-caused autofluorescence. Therefore, sections were incubated with alkaline phosphatase-conjugated anti-fluorescein (provided as Converter-AP in the In Situ Cell Detection Kit, AP - Roche Diagnostics GmbH, Mannheim, Germany). Incubation with the converter was performed overnight, at 4C. Afterwards, the sections were rinsed twice in TTBS (10 min) and once in TBS (10 min). Fast-Red tablet sets (Sigma Fast, Sigma Chemical Co., St. Louis, MO, USA) were used for signal development. The slides were mounted in Mowiol, and the preparations were evaluated under a Zeiss Axioskop (OPTON, Carl Zeiss, Gottingen, Germany) using DIC (Differential Interference Contrast) optics with visible and fluorescent illumination. The percentage of TUNEL-positive nuclei (TUNEL%) was calculated in 5 visual fields (400 magnification) of an individual sample. Photographs were taken on Kodak Gold 100 ASA (Eastman Kodak Co., Rochester, NY, USA) film with an MC80 Microscope Camera.

DNA exfraction and electrophoresis

Fat body lobes of Manduca sexta were collected at each day of the 5th larval stage (VO-V9). Collected samples were homogenized in order to extract total genomic DNA. The lysis buffer applied for isolation of DNA contained 150 mM NaCl, 25 mM Na-EDTA and 0.2% (w/ w) SDS (pH 8.0). Approximately 50 g tissue was lysed in 500 l lysis buffer. The lysate was treated with 5 l proteinase K (10 mg/ml) for 4 hours at 60C. DNA was extracted by standard phenol/chloroform extraction and precipitated with elhanol. Dried pellets were dissolved in 200 l TE buffer. Five l DNase-free RNase (2 mg/ml) was added to the samples and digestion proceeded for 30 min at 37C. The samples were reextracted by phenol/chloroform extraction and precipitated with ethanol. After washing the pellets with 70% ethanol they were finally dissolved in 50 l TE buffer. DNA was subjected to electrophoresis in 1.5% agarose gels and was visualized by ethidium bromide staining. Lambda DNA/HindIII Marker (MBI Fermentas, Vilnius, Lithuania) was used as size marker.

Antibodies

The rabbit antiserum to SDS-denatured ubiquilin/Ub-[gamma]- globulin conjugates was raised using the method described by Haas and Bright (1985) and was kindly provided by Dr. Fergus J. Doherty (Dept. of Biochemistry, University of Nottingham Medical School, UK).

The presence of proteasomes was tested employing different anti- bodies: polyclonal rabbit antiserum raised against the protcasome ATPase subunit (S6/MS73) using a recombinant protein expressed from pSMS73c (Dawson et al., 1995); polyclonal rabbit antiserum produced against purified human red blood cell-derived 20S proteasome proteolytic core particle (kind gift from Dr. Carlos Gorbea; Dept. of Biochemistry, University of Utah, US); polyclonal rabbit antiserum generated against purified recombinant Saccharomyces pombe S4 subunit of proteasome regulatory cap (also from Dr. Gorbea).

Immunoblotting

Homogenates of the fat body were prepared from larvae collected at the indicated time points; and proteins were separated by 10% SDS- PAGE, followed by blotting onto a sheet of Hybond ECL (0.2 m pore size) (Amersham Biosciences Ltd, Little Chalfont, England, UK) according to the method described by Towbin et al. (1979) using a Bio-Rad MiniBlot chamber (Bio-Rad Laboratories, Hercules, CA, USA).

Non-specific binding sites were blocked by 3% Carnation non fat dry milk powder dissolved in TPBS (Tris-huffcred saline (TBS) 0.15 M Tris-HCl, 0.5 M NaCl, pH 7.0, containing 0.5% Tween-20). After washing three times with TBS, parallel immunoblot analyses were carried out using polyclonal rabbit anti-Ub and anti-S6/MS73 antibodies.

Sheets were incubated overnight at 4C in the presence of primary antibody diluted 1:100 in TBS. Blots were washed with TBS and incubated in the presence of secondary antibody (AP-conjugated anti- rabbit antibody; Sigma ImmunoChemicals, St. Louis, MO, USA) diluted 1 : 1000 for 1 hour at room temperature. After final washes in TBS and TTBS blots were developed using freshly prepared NBT-BCIP (5bromo-4-chloro-3-indolyl phosphate/nitroblue telrazolium, Sigma Chemical Co., St. Louis, MO, USA) as substrate. Molecular weights were estimated using the SDS-PAGE Broad Range prestained standards (Bio-Rad Laboratories, Hercules, CA, USA).

Immunohistochemistry

The animals were fixed in Bouin fixative for 6 hours at room temperature and embedded in Paraplast as described above. Five-m serial sections were cut, placed on poly-L-lysine-coated slides and dried overnight at 39C. After deparaffination, rehydration and blocking (5% Carnation non-fat dry milk powder in TBS) sections were incubated overnight at 4 C in solutions of the primary antibodies (listed above), diluted 1 : 1000 (Ub); 1 :100 (20S); 1 : 1000 (84); 1 :500 (S6/MS73). Bound anti-proteasome antibodies were visualized using the EnVision System, HRP, Universal, Rabbit/Mouse (DAB) in accordance with the guidelines provided by the manufacturer (DakoCytomalion Denmark, Glostrup, Denmark).

Slides were mounted in Mowiol, and photographs were taken on Kodak Gold 100 ASA (Eastman Kodak Co., Rochester, NY, USA) film with a Zeiss Axioskop (OPTON, MC80 Microscope Camera, Germany) using DIC (Differential Interference Contrast) optics.

Caspase activity assay

M. sexta larvae were collected at each day of the 5th larval stage (V0-V9). Chilled caterpillars were dissected in TBS under a stereomicroscope to obtain fat body samples for caspase activity assays. The lobes of the fat body were sampled at random. The collected tissue samples were rinsed in a large volume (approximately 10 ) of ice-cold stabilization buffer (20mM HEPES; 1 mM EDTA; 10% sucrose; 10 mM dithiothreitol (DTT); pH 7.5) and were homogenized in a glass-Teflon homogenizer.

Tissue homogenates (500 l) were centrifuged al 12,000 rpm for 15 minutes. Clear, lipid-free supernatants were collected and their protein concentrations were determined by Bradford Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). The protein concentration of the samples was equalized by dilution with stabilization buffer. Twenty l stabilization buffer containing 20 mM Ac(N-acetyl)- DEVD(Asp-GluVal-Asp)-amc(7-amino-4-methylcouma rin) fluorogenic tetrapeptide substrate (BIOMOL Research Laboratories Inc., Plymouth Meeting, PA, USA) was added to 30 l protein homogenate. Incubation was carried out in 96-well microtiter plates in a final volume of 100 m stabilization buffer. DEVDase activity indicated by accumulation of fluorescent product (7-amino-4-methylcournarin) was measured (excitation 360 nm; emission 465 nm) at 10-min intervals for 1 hour (FL 600 Microplate Fluorescence Reader; Bio-Tek Instruments Inc., Winooski, VT, USA; with KC 4 KincliCalc tor Windows software).

Results

Morphological changes during the 5th larval stage

The morphology of the developing fat body cells was examined by light and electron microscopic techniques during the last larval stage in Manduca sezta. Apoptolic morphology (e.g. shrinkage of cells and nuclei, cytoplasmic blebbing, apoplotic bodies, ru\ptured mitochondria, condensation and marginalization of the chromalin in nuclei, or detached cells which lost their intercellular connections) was not observed neither by light nor by electron microscopic analysis in the fat body from the 5th larval stage.

Fig. 1. Light and electron microscopic analysis of fat body prepared from the 5th larval stage. The cells of fat body (trophocytes) are small in young feeding larvae (A: V1, azan; B: V2, haematoxylin and cosin), but their volume increases significantly with the amount of stored nutritive material in elder specimen. Arrows point to the eosinophilic protein storage granules of heterophagic origin appearing in the decomposing trophocytes coinciding with the onset of PCD (C: V4, haematoxylin and eosin) and remaining during the wandering period of the 5"' larval stage (D: V8, azan) Fb: fat body; Tr: trachea. The ultrastructure of the trophocytes shows an enlarged cytoplasm with a mass of stored nutritive material - glycogen and lipid - soon before the onset of autophagy (E: V2). Autophagosomes appeared in great numbers by the end of the feeding period (F: V4.5), indicating the commencement of the PCD process in these cells. Later on the autophagosomes fused with lysosomes forming the so-called autophagic vacuoles. Inside them the degradation of the sequestered organelles and materials began (G: V5). At the end of the last larval stage most of the cytoplasmic organelles were wrapped by autophagic vacuoles and the undigested residues turned into electron-dense material (H: V8). Note the nuclear morphology lacking the characteristic apoptotic features in any phase of this process. AV: autophagic vacuole; HR: endoplasmic reticulum; L: lipid droplet; N: nucleus. Bars: 25 m (A, B); 10 m (C, D); 1 m (E-H).

Fig. 1. Light and electron microscopic analysis of fat body prepared from the 5th larval stage. The cells of fat body (trophocytes) are small in young feeding larvae (A: V1, azan; B: V2, haematoxylin and cosin), but their volume increases significantly with the amount of stored nutritive material in elder specimen. Arrows point to the eosinophilic protein storage granules of heterophagic origin appearing in the decomposing trophocytes coinciding with the onset of PCD (C: V4, haematoxylin and eosin) and remaining during the wandering period of the 5th larval stage (D: V8, azan) Fb: fat body: Tr: trachea. The ultrastructurc of the trophocytes shows an enlarged cytoplasm with a mass of stored nutritive material - glycogen and lipid - soon before the onset of autophagy (E: V2). Autophagosomes appeared in great numbers by the end of the feeding period (F: V4.5), indicating the commencement of the PCD process in these cells. Later on the autophagosomes fused with lysosomes forming the so-called autophagic vacuoles. Inside them the degradation of the sequestered organelles and materials began (G: V5). At the end of the last larval stage most of the cytoplasmic organelles were wrapped by autophagic vacuoles and the undigested residues turned into electron-dense material (H: V8). Note the nuclear morphology lacking the characteristic apoptotic features in any phase of this process. AV: autophagic vacuole; ER: endoplasmic reticulum; L: lipid droplet; N: nucleus. Bars: 25 m (A, B); 10 m (C, D); 1 m (E-H).

The cells of the fat body are relatively small soon after the last larval ccdysis (V1) (Fig. 1A, B), but during the feeding period of the 5th larval stage the volume of the cells continuously increases. Large lipid droplets and areas containing glycogen appeared in the cells, and the volume of these structures increased subsequently, as the amount of stored fat and glycogen increased with age (Fig. 1E). Onset of significant structural changes was detected at V4, the last day of the feeding period, when autophagic vacuoles began to form in the trophocytes (Fig. 1C, D).

The first step in the formation of the autophagic vacuoles is the appearance of the so-called autophagosomes or segregosomes. One can more or less recognize the ultrastructure of the segregated organelles inside them (Fig. 1F). Later on, the autophagosomes fuse with lysosomes and the digestion of the macromolecules begins inside these organelles known as autophagic vacuoles. The autophagic vacuoles fuse very often with each other. The undigested residues appear on the electron micrographs as dark, electron-dense spots or smears inside them (Fig. 1G). At the end of the last larval stage the structure of the segregated organelles is totally destroyed. Only the undigested, homogeneously electron-dense material fills the inside of the large autophagic vacuoles (Fig. 1H). In this developmental phase most of the cytoplasm is wrapped by the autophagic vacuoles.

DNA fragmentation

The TUNEL (TdT-mediated dUTP Nick End Labeling) assay-one of the most commonly accepted techniques in the examination of apoptotic cell death - proved to be excellent for the in situ investigation of nuclei with strongly fragmented DNA in the fat body during the last larval stage of Manduca ,sexta. In order to quantify the TUNEL assay, the ratio of the TUNEL-positive cells (nuclei) was determined versus the total cell count (TUNEL%). The TUNEL assay was performed on cross-sections of Manduca sexta larvae, which were collected from each physiological period of the 5th larval stage as described in Materials and methods.

Neither during the feeding period (V1 and V2) (Fig. 2A) nor in the very early wandering period (V5) DNA fragmentation could be detected. The onset of DNA fragmentation was observed in the early phase of the wandering period at day 6 (V6) of the 5th larval stage (Fig. 2B). This result was confirmed by agarose gel electrophoretic analysis of DNA (see below). The sudden appearance of the TUNEL- positive nuclei in very high ratio at day V6 indicates a definite and synchronized process in this tissue. The cells with intense TUNEL staining were found in great numbers, and their spatial arrangement in the fat body of V6 and elder larvae showed a fairly scattered pattern, which excludes the possibility of necrotic cell death.

The ratio of TUNEL-positive cells was followed throughout the 5th larval stage and was plotted versus time. The graph of TUNEL% clearly showed that the percent of cell death showed a sharp increase in the fat body at (he early wandering period (V6). The relatively early DNA fragmentation corresponds to the sequence of apoptotic PCD described in earlier publications. The percent of TUNEL-positive nuclei remained at a relatively high level till the end of the 5th larval stage (Fig. 2C).

Agarose gel electrophoresis was performed in order to reveal DNA fragmentation as one of the most characteristic aspects of apoptotic processes. Total genomic DNA was extracted from samples of the fat body collected at each day of the 5th larval stage. DNA fragmentation did not occur during the feeding period, but a considerable fragmentation of DNA indicated by the appearance of low- molecular-weight fragments was observed in the samples from the wandering period (Fig. 2D).

Caspase activity

In order to explore the activity of executioner caspases during this normal developmental process we carried out an in vitro caspase activity assay applying a tetrapeptide substrate, DEVD-amc, designed for the CPP32 (human caspase-3) enzyme. The DEVD sequence, which derives originally from the PARP cleavage site, is also optimal for the related cystein proteases, primarily the other executioner caspases with short prodomain (caspase-7 and caspase-6). Therefore, this method can be used to study the general occurrence or absence of active caspases during this process. The activity of these enzymes was examined in the larval fat body homogenates. The samples of fat body were collected from each day of the 5"' larval stage of Manduca sexta.

As earlier publications described significant expression of caspase homologues in the mid gut and the salivary gland during Drosophila larval development (Dorstyn et al., 1999b; Harvey et al., 2001), we considered the protein extracts of larval mid gut of Manduca sexta a useful positive control in this assay.

The measured fluorescence values were normalized to 2.72 mg/ml nominal protein concentration. In contrast to the applied positive control, the obtained data from the measurement of the Tat body samples did not differ significantly from their initial values, indicating the obvious lack of the enzymatic activity (Fig. 3). For proper comparison, linear regression was done on the values belonging to the linear portion of the enzymatic reaction curve of the control and the obtained values from the fat body samples using the function SLOPE of Microsoft Excel 2000.

Fig. 2. Detection of DNA fragmentation in the fat body of Manduca xexla from the 5th larval stage. The light-microscopic in situ TUNEL analysis of larval fat body from feeding larvae did not reveal nuclei with fragmented DNA (A: V1, 100 ). Great numbers of TUNEL- positive nuclei indicated the widespread DNA fragmentation during the early phase of the wandering period (B: V6, 200 ). The protein storage granules accompanying the cellular decomposition were clearly shown nearby the nuclei. C: Ratio of TUNEL-positive nuclei versus time in the fat body. Note that DNA fragmentation is a rapid, intense process, which commences in the early wandering period (V6) and persists till the end of the 5th larval stage. D: Electrophoretic analyis of total genomic DNA isolated from fat body during the 5th larval stage. Appearance of low-molecular-weight DNA fragments at day 5 (V5) coincides with the onset of wandering behavior. Strong DNA fragmentation can be observed in the samples throughout the wandering period (V5-V8).

Ubiquitin (Ub)

Total protein homogenates prepared from the fat body of the larvae of Manduca sexta were collected from each day of the 5th larval stage and were analyzed by Western blot technique. The Westernblot profile of the samples demonstrated the lack of Ub throughout the feeding period, but the appearance of Ub was revealed in cells of the fat body at the commencement of the wandering period. Alteration of the ubiquitylated protein composition was not observed during this period, however the progressive Uh tagging was indicated in the wandering larvae by the conspicuous and increasing "ubiquitin smear" (data not shown).

Fig. 3. DEVD-amc-cleaving activity of the fat body cell extract. Enzymatic activity was measured in order to disclose the presence of the active executioner caspases during this period. The bars represent the slopes of kinetic curves of fat body samples collected from the 5th larval stage. Comparing to the applied control (mid gut cell hotnogenate), the DEVD-amc cleaving activity of the fat body extract remained on negligible level throughout the 5th larval stage.

Immunostaining of the larval fat body lobes by the anti-Ub antibody gave an unexpected result. During the feeding period of the last larval stage we could not detect the presence of the Uh in the fat body cells. But just before the beginning of the wandering period, when the first autophagic vacuoles appear in the cytoplasm, a characteristic localization of Ub was observed. It was not present in the cytoplasm or in the elements of the lysosomal compartment but the nuclei were stained. The reaction was not very strong using cither the alkaline-phosphatase or the FITC-conjugated second antibodies, but practically all of the nuclei were stained uniformly. This pattern of immunolocalization was observed continuously till the beginning of the pupal stage, when Ub disappeared again from the nuclei of the fat body cells (Fig. 4A, B).

Proteasome

Immunohistochemical investigation was performed for revealing the in situ localization of the 26S proteasome in the cells of the larval fat body collected from the feeding (V2) and the wandering period (V8/W3) of the last larval stage. Three components of the 26S proteasome were investigated: the 20S proteolytic core particle, as well as the S4 and the S6/MS73 subunits of the regulatory cap. Each of these components showed significant nuclear localization in both physiological periods. However, the immunostaining was much more intense in the wandering larvae, indicating the growing amount of the 26S proteasome. This result is in line with the above mentioned nuclear localization of Ub, showing the nuclei as the major working area ol the Uh-protcasome system in the fat body during this developmental phase (Fig. 4C-H).

The presence of the 26S proteasome in the larval fat body throughout the 5lh larval stage was also surveyed by immunoblotting using the anti-S6/MS73 antibody, which recognizes an ATPase subunit of the 26S proteasome regulatory cap. One distinct band with an apparent molecular mass of 48 kDa was labeled in the Western blot, which very likely is identical to the MS73 protein of Manduca sexta (47 kDa). The protein was not detected at the very early phase of the last larval stage (Vl ), but increasing amounts of MS73 appeared in the elder larvae indicating the greater accumulation of the 26S proteasome in this tissue (Fig. 5).

Discussion

The apoptotic PCD - the mitochondrial as well as the death receptor pathway - has been studied extensively in vertebrates, primarily in cultured murine and human cells. However, it is an open question whether these pathways exist and/or coexist during the physiological, developmental processes of insects. To approach this problem, we studied the events of PCD in the larval fat body cells during the last stage of larval development of Manduca sexta. This organ can be regarded as a practically homogeneous tissue, consisting of a single cell type, the trophocytes, which die via autophagocytosis (Locke and Collins, 1966; Sass and Kovacs, 1975, 1977).

We made an attempt to detect in these cells the morphologic features which are commonly regarded as hallmarks of apoptosis and studied changes in the location and activities of proteins known to be involved in the process of apoptotic cell death.

According to the earlier descriptions, accumulation of clumps of condensed chromatin frequently attached to the inner surface of the nuclear membrane is a typical morphologic indication of ongoing apoptosis. Later on the nuclei of these cells become fragmented, which is followed by extensive vacuolization, rupture of mitochondria, swelling of the endoplasmic reticulum cisternae and blebbing of the cell membrane (zeiosis). The specific shrinkage of the cells is the consequence of the cxocytosis of these vacuoles and pinching off the membrane-bound apoptotic bodies. Finally, the dying cell loses its intercellular junctions, detaches itself from its cellular environment, and becomes engulfed by phagocytes or normal adjacent cells (Kerr et al., 1972; Wyllie, 1980).

In this study we did not find any sign of the above described apoptotic morphology in the larval fat body. Disintegrated, fragmented or shrinking cells were not registered by light microscopic evaluation and the electron microscopic analysis did not reveal the characteristic nuclear morphology, the blebbing, or the apoptotic body formation. However, hemocytes, which can consume the cell debris were detected nearby the lobes of the fat body. On the other hand, extensive autophagy - indicated by the extremely quick and extensive increase in the lysosomal compartment of the cells - was observed in the larval fat body and the mid gut from the beginning of the wandering period confirming the results of earlier studies made on other Lepidopleran insects (Locke and Collins, 1966; Sass and Kovacs, 1975, 1977). This event definitely precedes the DNA fragmentation, which starts during the early phase (day 6) of the wandering period of the 5th larval stage in the fat body cells (see below).

Fig. 4. The Ub-proleasome system showed significant nuclear localization in the fat body as revealed by immunohistochemistry during the feeding (V2) and the wandering period (V8) of the 5th larval stage. Ub and the 26S proteasome accumulated in greater amounts in the nuclei in the wandering larvae. The presence of the 26S proteasome was investigated by several independent antibody probes targeting the protcolytic core particle (20S) and the proteasome regulatory cap (S6/MS73, S4). Ub staining was weak, but perceptible (A), 20S (C) and S6/MS73 (e) immunolabeling yielded definitely positive results; but the quantity of the S4 subunit (G) was negligible in the feeding larvae (V2). Each antibody probes (Ub, 20S, S6/MS73, S4) revealed greater accumulation of the Ub- proteasome system in the nuclei of trophocytes in the wandering animals (B, D, F, H), corresponding with the course of PCD. (A, B: 160 C-H: 400 )

Our observations are in agreement with earlier descriptions (Schwartz, 1992; Zakeri et al., 1993), showing that in the dying intersegmental muscles during the postembryonic development of insects the classical apoptotic morphology cannot be recognized. However, some other data suggest that the autophagic and apoptotic pathways may coexist in certain degrading larval organs of insects, e.g. in salivary and prothoracic glands (Dai and Gilbert, 1997).

The internucleosomal cleavage of genomic DNA by activated endonucleases has been thought to be responsible for the development of nuclear apoptotic morphology (Wyllie and Morris, 1982). Although, DNA fragmentation is now widely considered diagnostic for apoptosis it has been reported by some authors that cell death can occur in the absence of DNA cleavage (Schultze-Osthoff et al., 1994).

One of the aims of the present work was to see whether DNA fragmentation occurs in dying insect larval cells showing typical morphologic signs of autophagocytotic cell death. Our results prove that an early DNA fragmentation is observable at the beginning and during the metamorphic process in the larval fat body. This was confirmed by two independent methods, by the gel electrophoretic analysis of the total DNA extract of these tissues and by the TUNEL assay showing a high ratio of TUNEL-positive nuclei in the fat body of the wandering-period last larval stage.

The DNA fragmentation, as an integral part of the PCD in natural developmental processes of insects was described previously in those organs where the apoptotic and autophagic features were simultaneously observed but the performance of the classic DNA ladder failed (Zakeri et al., f 993) with the only exception of a highly synchronized in vitro cell culture triggered to death (Dai and Gilbert, 1999). According to our best knowledge it is the first report on the occurrence of DNA fragmentation in larval insect cells with morphological pure autophagic phenotype.

Fig. 5. Western blot analysis of the S6/MS73 subunit of the proteasome regulatory cap disclosed one single, specific band at 48 kDa from the protein extract of fat body. This protein was absent in the very young feeding animals (V1). The earliest appearance of S6/ MS73 takes place in the V2 feeding larvae, which is subsequently followed by greater accumulation of the protein.

The facts about apoptosis-related regulators in insect development can be summarized as follows. Although, several Bcl-2/ ccd-9 members were described in Drosophila, and for example the overexpression of the DROB-I gene promoted dramatic cell death in particular elements of the CNS (Brachmann et al., 2000; Boise et al., 1993), we were unable to obtain valuable data concerning the role of this protein family in the autophagocytotic process in the fat body. The reason for this is that the antibody developed against the mammalian Bcl-x protein does not give a specific reaction against its Drosophila orthologue on Western blots and immunohistochemistry.

The function of active caspases during the natural developmental process of insects is not clarified entirely. However ICE/ ced-3 gene \family members in Drosophila have been published in great numbers during the last few years, and it has been shown that cell death can be realized via the powerful caspase pathway by overexpression of these genes (Dorstyn et al., 1999a, b; Harvey el al., 2001). In our study the cleavage activity of the executioner caspases was measured by a caspase activity assay using a tetrapeptide substrate (Ac-DEVD-amc; Biomol). However, this fluorogenic substrate designed for human caspase-3 is also optimal for the related cysteine proteases, primarily the other executioner caspases (caspase-7 and caspase-6). Therefore, this assay can be used to study the general occurrence or absence of active caspases in a given biological system. Three caspases have been described in Drosophila so far with short prodomains, DCP-I, drICE, and DECAY, which are therefore considered likely to be downstream, executioner caspases. There is evidence for two of these - DCP-I (Song et al., 1997) and DECAY (Dorstyn et al.,1999b) - to be able to cleave at DEVD sites.

Our experiments concerning the presence of caspase-3 in the fat body gave negative results. A commercially available antibody developed against mammalian caspase-3 did not give a specific reaction in our experiments (not shown). Moreover, in the fat body of Manduca sexta a very low, negligible activity of DEVDase has been detected and the changes of this activity did not show any relationship with the metamorphic events during the last larval stage. Thus, we could not find evidence for the participation of caspase-3 in the degradation of larval fat body cells. Thus we were not able to identify apoptotic markers, apart from the fragmentation of DNA.

Several lines of evidence suggest that the commitment of intersegmental muscles of Manduca sexta to die does not involve metabolic shutdown but does involve selective regulation of gene expression. It has been shown that one of the upregulated genes before and during muscle degeneration is the polyubiquitin gene which has nine repeats of the ubiquitin coding sequence (Schwarlz. et al., 1990). The trigger for cell death and the dramatic increase in polyubiquitin expression in this system is the decline of the level of circulating molting hormone. It is well known that ubiquitin may serve as a macromolecular tag attached to certain proteins to mark them for degradation by the proteasome (Pickart, 2001). The ubiquitin-protein conjugates may also be decomposed in the lysosomal compartment of the cells (as it was described in various cell types) (Laszlo et al., 1990; Mayer et al., 1991).

Therefore, the expected sites of location of ubiquitin or the ubiquilin-protein complexes in the dying fat body cells were the cytoplasmic proteasomes and/or the elements of the lysosomal system, such as the multivesicular bodies and autophagic vacuoles. In contrast with these expectations we detected accumulation of anti- ubiquitin-positive material in the nuclei of the fat body cells just before and at the beginning of the autophagic processes. It is an interesting result since in a number of cases evidence showing that the ubiquitin-proteasome system may act as a key regulator of gene expression in cukaryotic cells has been reported. Ubiquitin regulates the function of RNA polymerase II not only by the destruction of gene-regulating proteins, but by mechanisms which appear to be independent of the proteasome (Conaway et al., 2002). It is well known that the onset of autophagy in the insect larval cells is triggered by the rise in ecdysone level (Sass and Kovacs, 1977, 1980). The nuclear location and accumulation of ubiquitin and proteasomes which coincide with the start of autophagy suggest that this highly conserved system may be involved in the regulation of differential gene activation necessary to accomplish the process of autophagocytosis in the larval fat body cells. Another possible reason for the nuclear accumulation of ubiquitin could be the increasing DNA fragmentation, as there is recent evidence that ubiquitylation and presumably destruction by proteasomes of the stalled RNA polymerase II molecules present at lesions of damaged DNA takes place (Lee et al., 2002).

The physiological or programmed cell death is a very complex and highly organized process involving several molecular events. Although the connections of these events are barely explored, several alternative regulation mechanisms are recognized and described, like apoptosis, autophagocytosis or proteasomal degradation. Apoptosis, based on the crucial caspase cascade, is the most investigated, but not the only cell death mechanism. The significance of the so-called caspascindependent cell death forms, like autophagocytosis or proteasomal digestion, is understood better and better. Considering these, some authors emphasize a more comprehensive attitude in the evaluation of cell death processes (Lockshin and Zakeri, 2002).

Summarizing our data we consider the degradation process in the larval fat body cells during the post-embryonic development of Manduca sexta as a programmed cell death with typical autophagic character. We did not find any substantial evidence for the presence of features characterizing the apoptotic cell death - apart from the DNA fragmentation. The correlation of the latter with autophagy needs further investigation. Our results show that the autophagic process is accompanied by increased levels of ubiquitin found in the nucleus which thus seems to be a characteristic molecular event signaling the onset of autophagy in the Manduca sexta larval fat body cells.

In conclusion, in the larval fat body cells the molting hormone induces only the autophagic mechanism of PCD and therefore it represents a good model system to study the hormonal and genetic regulation and the molecular mechanism of autophagocytosis in pure form.

Acknowledgements. We thank Karolyne Valoczy, Mariann Sarody and Sarolta Sipos for skillful assistance. This work was supported by the Hungarian Ministry of Education under Grant MEDICHEM 1/047 NKFP provided to M. Sass.

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Ferenc Muller1), Csaba Adori, Miklos Sass

Department of General Zoology, Eotvos Lorand University, Budapest, Hungary

Received September 25, 2003

Received in revised version December 15, 2003

Accepted January 19, 2004

1) Corresponding author: Dr. Ferenc Muller, Department of General Zoology, Eotvos Lorand University, Budapest, P.O. Box 120, H-1518 Budapest, Hungary, e-mail: fefe@cerberus.elte.hu, Fax: +36 1 381 2184.\

Copyright Urban & Fischer Verlag Mar 2004

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