InteractiveFly: GeneBrief

Dynamin related protein 1: Biological Overview | References

The role of mitochondria in Drosophila programmed cell death remains unclear, although certain gene products that regulate cell death seem to be evolutionarily conserved. This study found that developmental programmed cell death stimuli in vivo and multiple apoptotic stimuli ex vivo induce dramatic mitochondrial fragmentation upstream of effector caspase activation, phosphatidylserine exposure, and nuclear condensation in Drosophila cells. Unlike genotoxic stress, a lipid cell death mediator induces an increase in mitochondrial contiguity prior to fragmentation of the mitochondria. Dynamin related protein 1 (Drp1), is important for mitochondrial disruption. Using genetic mutants and RNAi-mediated knockdown of drp-1, it was found that Drp1 not only regulates mitochondrial fission in normal cells, but mediates mitochondrial fragmentation during programmed cell death. Mitochondria in drp-1 mutants fail to fragment, resulting in hyperplasia of tissues in vivo and protection of cells from multiple apoptotic stimuli ex vivo. Thus, mitochondrial remodeling is capable of modifying the propensity of cells to undergo death in Drosophila (Goyal, 2007).

Programmed cell death (PCD) plays an important role in sculpting tissues during animal development. The molecular regulators that are central to this process seem to be evolutionarily conserved from worms to mammals and include autocatalytic initiator caspases, trans-activable effector caspases, cytosolic activating factors (APAF-1), and multidomain Bcl-2 proteins. The proapoptotic Bcl-2-family proteins oligomerize and permeabilize mitochondria, releasing intermembrane space components such as cytochrome-C and Smac/DIABLO into the cytosol, where they activate initiator caspases by an ATP-dependent mechanism. Initiator caspases trans-activate effector caspases that cleave multiple cellular substrates, resulting in DNA degradation, nuclear condensation, and loss of cell integrity (Goyal, 2007 and references therein).

Mitochondrial outer-membrane permeabilization has been proposed to depend on the mitochondrial fission and fusion machinery (Perfettini, 2005; Youle, 2005). Consistent with this, mitochondria undergo dramatic fragmentation very close in time to cytochrome-C release during mammalian cell death (Frank, 2001; Mancini, 1997). Furthermore, an increase in mitochondrial fragmentation and a block in mitochondrial fusion are essential for cell death progression (Frank, 2001; Karbowski, 2002; Yu, 2005). In normal cells, the balance in the rates of mitochondrial fission and fusion regulated by Dynamin-related protein-1 (Drp-1), Fis-1 and endophilin (fission), or Mitofusins and Opa-1 (fusion) maintains the dynamic, interconnected mitochondrial tubules (Meeusen, 2005; Okamoto, 2005; Yaffe, 1999). An increase in recruitment of Drp-1 to the mitochondria accentuates staurosporine, lipid, and free oxygen radical stress-induced mitochondrial outer-membrane permeabilization (Breckenridge, 2003; Szabadkai, 2004). Moreover, multiple apoptotic stimuli induce mitochondrial recruitment of the proapoptotic Bcl-2-family protein, Bax, to Drp-1 and Mitofusin-2-positive putative mitochondrial fragmentation sites (Karbowski, 2002; Neuspiel, 2005) in a Fis-1-dependent manner (Lee, 2004), consistent with a role for mitochondrial fission and fusion machinery in cell death (Goyal, 2007).

In Drosophila, RHG-family proteins (Reaper, Hid and Grim), genotoxic stresses, and protein synthesis inhibitors antagonize Drosophila inhibitor of apoptosis protein-1 (DIAP-1)-mediated inhibition of the activation of the apical caspase Dronc in an ARK- (Drosophila APAF-1) and ATP-dependent manner, leading to effector caspase activation and cell death. The role of mitochondria in this process is unclear. Cytochrome-C has been shown to be differentially displayed from the mitochondria during cell death. Knockdown of Drosophila cytochrome-C did not affect cell death triggered by genotoxic stress in vitro and ex vivo or developmental stimuli in vivo, although certain nonapoptotic caspase activation pathways utilized during sperm individualization were affected. Furthermore, mitochondrial morphology during Drosophila PCD has not been previously reported (Goyal, 2007 and references therein).

This study shows that multiple apoptotic stimuli result in mitochondrial fragmentation upstream of caspase activation, phosphatidylserine exposure, and nuclear condensation in Drosophila cells. While etoposide induced mitochondrial fragmentation, C₆-ceramide resulted in an increase in mitochondrial contiguity prior to its fragmentation. drp-1 mutant or RNAi-treated S2R⁺ cells are considerably protected from multiple apoptotic stimuli, consistent with reduced mitochondrial fragmentation. Thus, mitochondrial remodeling plays an important role in modifying the propensity of cells to undergo PCD in Drosophila (Goyal, 2007).

Precisely timed ecdysone pulses induce Reaper and Hid expression in the Drosophila larval midgut (0 hr after puparium formation [APF]) or the salivary gland (10 hr APF) and trigger developmental PCD. Mitochondria, visualized by using matrix-targeted GFP (Mito-GFP) in acridine orange-positive, dying prepupal midgut cells (1 hr APF) and salivary glands (minus 4 hr APF), are remarkably fragmented, unlike third-instar larval (-4 hr APF) mitochondria. Quantification revealed a dramatic decrease in the prepupal mitochondrial cross-sectional area (CSA; midgut and salivary gland and a significant increase in the number of mitochondria per cell. Moreover, ecdysone-induced mitochondrial fragmentation is mimicked ex vivo on third-instar larval wing discs by using 1 mM ecdysone for 2 hr. In addition, overexpression of Hid resulted in mitochondrial fragmentation in acridine orange-positive eye disc cells. Thus, mitochondria in Drosophila tissues fragment during PCD, as has been reported in C. elegans (Jagasia, 2005) and mammalian cells (Frank, 2001; Goyal, 2007).

To assess the role of mitochondrial remodeling in PCD, mitochondrial morphology was temporally characterized in etoposide-, actinomycin-D-, cycloheximide-, or C₆-ceramide (a lipid cell death mediator)-treated larval hemocytes and the S2R⁺ cell line. A 3- to 4-fold increase in nuclear condensation (6 hr) was preceded by effector caspase activation (5 hr) and phosphatidylserine (PS) exposure in propidium iodide (PI)-negative hemocytes (6 hr). These cells subsequently (10 hr) became characteristically blebbed and PI permeable. The number of etoposide-treated apoptotic hemocytes increased with time. Interestingly, mitochondrial fragmentation, as confirmed by quantifying functionally isolated mitochondria at 3 hr, preceded the onset of PS exposure or nuclear damage. Quantification showed an increase in the number of mitochondria and the contribution of fragmented mitochondria to the mitochondrial cross-sectional area (CSA). Mitochondrial fragmentation was also observed in cycloheximide- or actinomycin-D-treated, Mito-YFP-transfected S2R⁺ cells (Goyal, 2007).

Surprisingly, mitochondria in C₆-ceramide-treated (30-60 min) hemocytes that had normal nuclei were highly contiguous. Quantifying functionally isolated mitochondrial CSA per cell showed a significant increase in the contribution of tubular or extensively tubular mitochondria in these cells when compared with untreated cells. However, by 4 hr, these extensively tubular mitochondria underwent fragmentation in FITC-Annexin V (AnV)-negative hemocytes that had normal nuclei , similar to what was observed with genotoxic stress (Goyal, 2007).

Therefore, genotoxic stresses trigger mitochondrial fragmentation, while the lipid cell death mediator induces increased mitochondrial contiguity and subsequent fragmentation prior to phosphatidylserine exposure, nuclear condensation, and finally plasma membrane permeability during Drosophila cell death (Goyal, 2007).

In hemocytes incubated with an apoptotic stimulus, mitochondrial fragmentation (3-4 hr) preceded any detectable effector caspase activation. Furthermore, inhibiting caspases with zVAD-fmk or by overexpressing DIAP-1 (DIAP-1+) did not affect mitochondrial fragmentation, although hemocyte death was inhibited, as revealed by a lack of apoptotic markers. In addition, overexpression of Dcp-1, a Drosophila effector caspase, did not affect mitochondrial morphology. Thus, mitochondrial fragmentation is upstream of effector caspase activation (Goyal, 2007).

The drp-1 mutants used to study the role of mitochondrial remodeling during Drosophila PCD are functional null alleles, drp-1² (Gly293Ser mutation), picked in a forward screen for genes affecting neurotransmission and drp-1^{[KG 03815]}, a P element insertion between the first two exons of drp-1 (13510 in this study) and a hypomorph, nrd^D46 (Arg278Trp mutation; 3665 in this study) (Rikhy, 2007; Verstreken, 2005). drp-1², 13510, and the deficiency Df Exel6008 were second-instar larval lethal; however, drp-1² yielded bang-sensitive escapers (Verstreken, 2005). The hypomorphic trans-allelic combination of 3665/13510 was third-instar larval lethal, although it yielded a few temperature-sensitive adults (Rikhy, 2007). A genomic duplication of drp-1 (Dp [2;1] JS13 [Rikhy, 2007]) completely rescued the lethality associated with drp-1², 13510, and 3665/13510 (Goyal, 2007).

Mitochondria in drp-1² and 3665/13510 hemocytes were extensively tubular when compared with wild-type mitochondria. Quantifying mitochondrial morphology revealed a 2-fold decrease in the number of mitochondria and a significant increase in the contribution of tubular and extensively tubular mitochondria to the total mitochondrial CSA in drp-1 mutant hemocytes when compared with wild-type cells. Interestingly, 13510/+ hemocytes or eye disc cells displayed a dominant mitochondrial fission defect that was completely rescued by a genomic duplication of drp-1. The mitochondrial fission defect in mutant cells could result from a reduced mitochondrial association of Drp-1 (Goyal, 2007).

An increase in mitochondrial contiguity due to a loss of Drp-1 function was also confirmed by measuring fluorescence recovery after photobleaching (FRAP) of Mito-YFP in drp-1 RNAi-treated S2R⁺ cells that had extensively tubular mitochondria. Relative FRAP of Mito-YFP in a defined mitochondrial region in drp-1 RNAi-treated cells was significantly higher than that observed in mock RNAi-treated cells (Goyal, 2007).

drp-1 mutant hemocytes were protected from etoposide-induced death up to at least 10 hr, as revealed by a lack of caspase activation, PS exposure, or PI permeability in the majority (~80%) of these cells. Furthermore, drp-1 mutant and dsRNA-treated S2R⁺ cells were significantly protected from cycloheximide-, actinomycin-D-, or UV-B-induced death. Consistent with increased protection, mitochondria in the majority (~98%) of etoposide-treated drp-1² hemocytes failed to fragment. Interestingly, mitochondria in etoposide-treated 3665/13510 hemocytes revealed a tubular, yet beaded and swollen intermediate in mitochondrial fragmentation by 4 hr that yielded some fragmented mitochondria in few (~25%) cells later. Therefore, reduced (drp-1²) or delayed (3665/13510) mitochondrial fragmentation decreased effector caspase activation and protected cells from genotoxic stress. Moreover, an increase in expression of Drp-1 in hemocytes resulted in enhancement of etoposide-induced cell death (Goyal, 2007).

The majority (~70%) of the C₆-ceramide-treated drp-1² hemocytes did not show effector caspase activation or PS exposure and displayed significant protection, similar to what was observed with etoposide, although hemocytes derived from the weaker allelic combination, 13510/3665, were apoptotic. Unlike 13510/3665 mitochondria, drp-1² mitochondria failed to fragment, consistent with an essential role for Drp-1-mediated mitochondrial fragmentation during apoptosis in Drosophila. Moreover, developmental PCD in drp-1² mutant larvae was considerably reduced, as revealed by the enlarged central nervous system and a prominently elongated ventral ganglion, similar to other PCD-defective mutants reported (Mills, 2006; Goyal, 2007).

During metamorphosis, the first ecdysone pulse triggers mitochondrial fragmentation in prepupal tissues, although it is after the second ecdysone pulse that salivary gland histolysis occurs. It is likely that DIAP-1 inhibits caspases in these cells that have fragmented mitochondria until it is downregulated at the transcriptional level or degraded after the second ecdysone pulse (Yin, 2005). Interestingly, this was mimicked ex vivo in etoposide-treated DIAP-1+ hemocytes (Goyal, 2007).

The data presented in this study show involvement of mitochondrial fragmentation for ARK-mediated Dronc activation during cell death. The RHG-family proteins that localize to the mitochondria (Claveria, 2002; Haining, 1999; Olson, 2003) might activate Drp-1-mediated mitochondrial fragmentation. This could result in exposure of cytochrome-C (Varkey, 1999) or release of Peanut (Gottfried, 2004), which antagonize DIAP-1-mediated suppression of Dronc. However, since Drosophila PCD is unaffected upon knockdown of cytochrome-C (Dorstyn, 2004), mitochondrial fragmentation in Drosophila and mammalian cells would increase mitochondrial surface area and perhaps the concentration of bulky head group lipids on the outer mitochondrial membrane, facilitating recruitment of proapoptotic proteins. Drp-1 might organize sites for Drosophila Bcl-2-family protein Debcl function on mitochondria (Dorstyn, 2002) that are similar to mitochondrial sites of Bax recruitment in mammalian cells (Karbowski, 2002; Goyal, 2007 and references therein).

These results provide the first evidence that Drp-1-mediated mitochondrial fragmentation upstream of effector caspase activation modifies apoptotic sensitivity. Thus, mitochondrial fragmentation, like caspase activation, plays a conserved and unifying role in diverse cell death pathways from worms to mammals (Frank, 2001; Jagasia, 2005). Although the function of the highly contiguous mitochondria during lipid-induced cell death remains poorly understood, this study brings to the forefront a modulatory role for mitochondrial remodeling in determining the susceptibility of Drosophila cells to death.

Mitochondrial disruption in Drosophila apoptosis

Mitochondrial disruption is a conserved aspect of apoptosis, seen in many species from mammals to nematodes. Despite significant conservation of other elements of the apoptotic pathway in Drosophila, a broad role for mitochondrial changes in apoptosis in flies remains unconfirmed. This study shows that Drosophila mitochondria become permeable in response to the expression of Reaper and Hid, endogenous regulators of developmental apoptosis. Caspase activation in the absence of Reaper and Hid is not sufficient to permeabilize mitochondria, but caspases play a role in Reaper- and Hid-induced mitochondrial changes. Reaper and Hid rapidly localize to mitochondria, resulting in changes in mitochondrial ultrastructure. The dynamin-related protein, Dynamin related protein 1 (Drp1), is important for Reaper- and DNA-damage-induced mitochondrial disruption. Significantly, it was shows that inhibition of Reaper or Hid mitochondrial localization or inhibition of Drp1 significantly inhibits apoptosis, indicating a role for mitochondrial disruption in fly apoptosis (Abdelwahid, 2007).

A role for mitochondria in apoptosis appears to be conserved from mammals to nematodes to yeast. The lack of clear evidence that mitochondria play a role in Drosophila apoptosis has prompted discussion of whether flies represent an evolutionary outlier in this highly conserved process. The data strongly suggests that mitochondrial disruption also plays a role in Drosophila apoptosis (Abdelwahid, 2007).

The data show that mitochondria rapidly become permeable to Cyt c when Rpr or Hid are expressed, both in cultured cells and in vivo. This alteration in mitochondrial permeability was also seen during DNA-damage-induced apoptosis. Importantly, it was demonstrated that the mitochondrial permeabilization during DNA-damage-induced apoptosis is dependent on the genes in the H99 interval. Taken together, these data indicate that Rpr and Hid are both necessary and sufficient for mitochondrial permeabilization (Abdelwahid, 2007).

In contrast, apoptosis induced by Actinomycin D, UV, and DIAP1 RNAi does not result in mitochondrial permeabilization. This indicates that caspase activation alone is not sufficient to induce mitochondrial permeabilization and that the mitochondrial permeabilization seen on Rpr or Hid induction is not simply a general late event in apoptosis. The efficient cell killing by Actinomycin D, UV, and DIAP1 RNAi also implies that mitochondrial permeabilization is not important for all apoptosis in Drosophila cells. Rather, it suggests that the Rpr and Hid proteins have a specific activity on the mitochondria that results in mitochondrial permeabilization to execute apoptosis in a timely manner (Abdelwahid, 2007).

The effects of Rpr and Hid on mitochondria were not limited to permeabilization. It was found that mitochondrial morphology is dramatically altered within 90 min of Rpr or Hid expression, in both S2 cells and embryos. A variety of defects were found in mitochondrial ultrastructure ranging from a rounded appearance, to bulging (and occasional rupture) of the outer mitochondrial membrane, to swelling of the matrix and disruption of the cristae. This was rarely seen with other inducers of apoptosis. Rpr and Hid may directly cause altered mitochondrial morphology or could act indirectly through other proteins localized at the mitochondria (Abdelwahid, 2007).

The absence of mitochondrial permeabilization in cells treated with DIAP1 dsRNA indicates that the mitochondrial function of Rpr and Hid is independent of their ability to inhibit DIAP1. This is confirmed by data showing that expression of DeltaN-Rpr results in mitochondrial permeabilization despite the fact that this protein lacks the necessary motif to inhibit DIAP1 antiapoptotic activity. Taken together, these data demonstrate that Rpr and Hid have dual activities in the cell, both to inhibit DIAP1 and to permeabilize mitochondria. Data from other labs have suggested that Rpr is a multifunctional protein (Thomenius, 2006). The data confirm that Rpr has multiple proapoptotic activities in the fly (Abdelwahid, 2007).

The dual functionality of Rpr and Hid parallel the recently described role of C. elegans Egl-1 in mitochondrial damage (Jagasia, 2005). Egl-1 induces apoptosis by binding to Ced-9 to promote both the activation of the caspase Ced-3 and mitochondrial fragmentation. Similarly, Rpr and Hid bind to DIAP1, displacing active caspases and act on mitochondria to promote mitochondrial disruption. One difference between C. elegans and flies appears to be the requirement for caspase activity in the mitochondrial disruption. In C. elegans, Ced-3 is not required for fragmentation but is required for apoptosis in response to fragmentation. In Drosophila, caspase activity participates in the mitochondrial changes (Abdelwahid, 2007).

Two lines of evidence support a role for mitochondrial disruption in Drosophila apoptosis. First, Rpr and Hid must localize to mitochondria to elicit a full apoptotic response. Second, if mitochondrial disruption is blocked by inhibiting Dynamin related protein 1 (Drp1) expression, a decrease is seen in apoptosis. These data clearly indicate that mitochondrial localization of Rpr and Hid is required for a full apoptotic response in S2 cells. This agrees with previous data on Rpr and also with studies on a Grim mutant lacking a mitochondrial localization signal (Claveria, 2002; Olson, 2003; Chen 2004). Mitochondrial localization of Hid has been demonstrated in a heterologous system (Haining, 1999). In the Haining study, Hid killing was not compromised in the absence of mitochondrial localization, in contrast to the current observations in Drosophila cells. A role for mitochondrial localization is also supported by the finding that two mutant forms of Hid that lack mitochondrial localization in mammalian cells behave as weak loss-of-function alleles in the fly (Abdelwahid, 2007).

The mitochondrial fission protein Drp1 is implicated in mitochondrial disruption during apoptosis in yeast, nematodes, and mammals. The current data indicate a role for this protein in Rpr-induced and DNA-damage-induced mitochondrial disruption in S2 cells and in the embryo. Furthermore, the inhibition of mitochondrial disruption after Drp1 knockdown is correlated with a decrease in apoptosis, strongly suggesting that mitochondrial disruption contributes to the apoptotic response. It is interesting to note that Drp1 plays a conserved role in apoptosis in a wide variety of organisms but seems to function downstream of different pathways. In mammals, inhibition of Drp1 blocks apoptosis in response to activation of proapoptotic Bcl-2 family members. In C. elegans, Drp1 inhibition blocks endogenous death downstream of Egl1 and Ced9, also Bcl-2 family proteins (Jagasia, 2005). Even in yeast, the role of Drp1 in cell death can be modulated by Bcl-2 family proteins. Surprisingly, in flies, Drp1 appears to be acting downstream of a different family of apoptosis inducers, the RHG proteins. It remains to be seen whether a role for the fly Bcl-2 family proteins can be established in mitochondrial disruption (Abdelwahid, 2007).

Release of apoptogenic factors, most notably Cyt c, from the mitochondria is an essential step in most apoptosis in mammalian systems. However, the current work confirms the findings of others that Cyt c, although released from mitochondria by Rpr and Hid, is not important for Rpr or Hid killing. It should be noted that Cyt c has been shown to be important in some Drosophila developmental apoptosis. In these deaths, Hid is likely to act upstream of Cyt c release. If Cyt c release is required in some cells for Hid-mediated caspase activation, why not in S2 cells? It is possible that there are both Cyt c-dependent and -independent mechanisms for activating caspases, and these may be cell-type dependent. Recent data from mice carrying a nonapoptogenic form of Cyt c supports this model (Hao, 2005), since this study suggests that there is both Cyt c-dependent and -independent apoptosis during mouse development (Abdelwahid, 2007).

If release of Cyt c is not an essential step in apoptosis in most fly cells, is another apoptosis-inducing factor released during mitochondrial disruption? In mammalian cells, release of other mitochondrial proteins such as SMAC/Diablo, Omi/HTRA2, and AIF are proposed to contribute to apoptosis (Danial, 2004). There is some evidence that released mitochondrial factors do not contribute to caspase activation in the fly. Unlike in the mammalian system, mitochondrial lysates cannot activate caspases in fly cytoplasmic lysates (Means, 2006). An alternative possibility is that mitochondrial disruption per se might contribute to apoptosis in the fly through inhibition of normal mitochondrial functions essential for cell viability. This might serve as a backup system, to maximize apoptosis in cells that express low levels of the RHG proteins. A similar role for mitochondrial disruption (Rolland, 2006) has been proposed in C. elegans (Abdelwahid, 2007).

In sum, it is concluded from these studies that Drosophila is not an outlier in evolution with regard to the involvement of mitochondria in the apoptotic process. Rather, the data indicate that mitochondrial changes contribute to Drosophila apoptosis. The findings suggest that the view of the role of mitochondria in cell death has to be broadened beyond the release of proapoptotic factors, to include the general disruption of mitochondria, ensuring that doomed cells have no chance of recovery. Such a model would fit not only the changes seen in mammalian mitochondria, but also those found in yeast, C. elegans, and flies as well (Abdelwahid, 2007).

Roles for Drp1, a dynamin-related protein, and Milton, a Kinesin-associated protein, in mitochondrial segregation, unfurling and elongation during Drosophila spermatogenesis

Mitochondria undergo dramatic rearrangement during Drosophila spermatogenesis. In wild type testes, the many small mitochondria present in pre-meiotic spermatocytes later aggregate, fuse, and interwrap in post.meiotic haploid spermatids to form the spherical Nebenkern, whose two giant mitochondrial compartments later unfurl and elongate beside the growing flagellar axoneme. Drp1 encodes a dynamin-related protein whose homologs in many organisms mediate mitochondrial fission and whose Drosophila homolog is known to govern mitochondrial morphology in neurons. The milton gene encodes an adaptor protein that links mitochondria with kinesin and that is required for mitochondrial transport in Drosophila neurons. To determine the roles of Drp1 and Milton in spermatogenesis, the FLP-FRT mitotic recombination system was used to generate spermatocytes homozygous for mutations in either gene in an otherwise heterozygous background. It was found that absence of Drp1 leads to abnormal clustering of mitochondria in mature primary spermatocytes and aberrant unfurling of the mitochondrial derivatives in early Drp1 spermatids undergoing axonemal elongation. In milton spermatocytes, mitochondria are distributed normally; however, after meiosis, the Nebenkern is not strongly anchored to the nucleus, and the mitochondrial derivatives do not elongate properly. This work defines specific functions for Drp1 and Milton in the anchoring, unfurling, and elongation of mitochondria during sperm formation (Aldridge, 2007).

In order to define the roles for the essential genes Drp1 and milton in mitochondrial morphogenesis during Drosophila spermatogenesis, mosaic males were generated in which some spermatocytes became homozygous for mutant alleles. Homozygosity was indicated by loss of fluorescence associated with Ubi-GFP, originally expressed in heterozygous cells from the chromosome homologous to that carrying the mutation. Haploid spermatids derived from meiotic division of homozygous mutant spermatocytes were also marked by lack of fluorescence and were used to determine the post-meiotic roles of Drp1 and milton (Aldridge, 2007).

For most genes, the genotype of a pre-meiotic spermatocyte dictates the phenotype of any haploid spermatid derived thereof, regardless of which allele the spermatid inherits. Primary spermatocytes undergo a period of extensive pre-meiotic transcription during which the mRNAs for most of the genes required for post.meiotic spermatid differentiation are transcribed. Many of these messages undergo translational repression until the times when the gene products are needed during spermiogenesis. Post-meiotic spermatids are therefore mostly dormant transcriptionally but very active translationally. Spermatids derived from spermatocytes heterozygous for a loss of function allele typically still contain wild type mRNA and/or protein and are phenotypically normal, even if the particular spermatid has only the mutant allele. This idea applies to the expression of Ubi-GFP as well; all spermatids, even those carrying only the CyO second chromosome, in the testes of Ubi-.GFP/CyO heterozygous males have nuclear fluorescence. Conversely, for mutant recessive alleles of genes, spermatids will show a mutant spermiogenesis phenotype only if derived from a homozygous mutant spermatocyte. This approach for assessing the roles of Drp1 and milton in spermatocytes and spermatids is valid since homozygous mutant spermatocytes were generated simultaneously lacking Ubi-GFP and either Drp1 or Milton, and since phenotypes of the haploid spermatids derived through meiotic divisions of those cells could be subsequently characterized (Aldridge, 2007).

Since both Drp1 and milton are transcribed starting in primary spermatocytes after the gonial mitotic divisions but before meiosis, the mutant clones were generated at a stage prior to the expression of any gene product; therefore, perdurance of Drp1 or Milton in mutant clones is not a significant consideration. Furthermore, the alleles of Drp1 and milton with which were made homozygous germline clones are null or strong loss of function alleles. Drp1^KG03815 is a P element insertion in the first intron of Drp1 that causes lethality when homozygous and which fails to complement other lethal Drp1 alleles. The milt₉₂ allele contains a two base pair deletion in the coding region, and the resulting frame shift leads to a truncated Milton protein of only one third the normal length (Stowers, 2002). Phenotypes seen in milt₉₂ germline clones are solely due to the milt₉₂ mutation, since homozygous milt₉₂ FRT40A males carrying an extra wild type copy of milton are viable and fertile with normal spermatogenesis (Aldridge, 2007).

The phenotype of mutant Drp1 primary spermatocytes is consistent with a role for Drp1 in mitochondrial fission and suggests that mitochondrial fusion and fission are normally active and counterbalanced in primary spermatocytes, as in S. cerevisiae. After gonial mitotic divisions and before meiosis, primary spermatocytes grow dramatically in size. At an early stage during this process, mitochondria temporarily aggregate before dispersing again and multiplying. While previous studies have not indicated mitochondrial fusion and fission during the primary spermatocyte stage, the abnormal retention of a tight cluster of mitochondria in mature primary spermatocytes lacking Drp1 is consistent with the possibility that (1) this cluster represents fused mitochondria that cannot divide; (2) mitochondria normally fuse in spermatocytes, perhaps on a constant basis, and especially during the early 'polar' spermatocyte stage when mitochondria aggregate; and (3) in wild type cells, active Drp1-mediated division is required to balance fusion and to separate the mitochondrial network into the individual organelles seen in mature primary spermatocytes, reported to number 150 per medial cross section. While the mitochondrial fusion mediator Fzo is detectable only after meiosis, its paralog dMfn24 is likely mediating low-level mitochondrial fusion in pre-meiotic spermatocytes. In the absence of Drp1, fusion predominates and results in large mitochondrial conglomerations. The possibility that other subcellular structures may also be included in the mitochondrial clusters cannot be ruled out. The data are consistent with the abnormal clustering of mitochondria seen in the cell bodies of Drp1 mutant neurons (Aldridge, 2007).

The defect observed in Drp1 spermatocytes suggests that Drp1 is required for mitochondrial morphology at an early point in spermatogenesis. Failure of putative Drp1-mediated mitochondrial fission in Drp1 mutant primary spermatocytes, and the resulting formation of an interconnected mitochondrial mass, has serious implications for the segregation of mitochondria during subsequent meiotic divisions. In wild type testes, individual mitochondria align on the meiotic spindle during each meiotic division, enabling roughly even mitochondrial distribution to daughter cells. If the mitochondrial material within a primary spermatocyte comprises an indivisible mass, then such segregation of mitochondria to secondary spermatocytes and then to spermatids should prove difficult, unless the force of cytokinetic division can trigger the breakage of the mitochondrial mass spread between daughter cells. However, the nature of meiotic cytokinesis makes such forced mitochondrial breakage unlikely, since cytoplasmic bridges between the meiotic products of a primary spermatocyte remain open throughout spermatid differentiation (Aldridge, 2007).

The configuration of mitochondria in early spermatids derived from homozygous Drp1 spermatocytes indeed suggested that the mitochondrial material began as an indivisible mass, which could not be divided properly during meiotic cytokinesis. The mitochondrial material in up to four cells at a time appeared connected, passing through the cytoplasmic connections between spermatids. In spermatid cysts whose cytoplasmic connections had been broken open by the pressure of the cover slip to give a syncytial appearance, the mitochondrial masses still appeared connected. Some mutant spermatids appeared to lack mitochondria, perhaps as a result of meiotic divisions in which the entire mitochondrial mass was segregated by chance to one of the other meiotic products of the original spermatocyte. The data suggest that Drp1 has a conserved role in mitochondrial distribution during meiosis, since the Drp1 homolog Dnm1p is required for proper mitochondrial distribution during meiosis and sporulation in the budding yeast S. cerevisiae (Otsuga, 1998; Bleazard, 1999; Aldridge, 2007).

Drp1 is required not only for mitochondrial segregation during meiosis but also perhaps for mitochondrial unfurling during axoneme elongation. In wild type cells, the two mitochondrial derivatives within a Nebenkern disentangle from each other, with the large surface area of each mitochondrial derivative immediately stretched and elongated beside the growing flagellar axoneme. It is hypothesized that Drp1-mediated mitochondrial fission is required for the breaking of multiple topological links between the two mitochondrial derivatives within the Nebenkern during unfurling. Given that Drp1 cells are defective prior to and immediately after meiosis, the possibility cannot be definitively rule out that the observed unfurling defects are a secondary effect of the earlier phenomena; however, secondary effects on mitochondrial unfurling are not seen in other known mutants with early mitochondrial defects. For example, an abnormally large Nebenkern initially associated with four nuclei (resulting from a meiotic cytokinesis defect) in four wheel drive mutants properly unfurls and elongates. Furthermore, in parkin and pink1 mutants, whose Nebenkerne consist of one compartment rather than two, mitochondrial unfurling still leads to a cohesive, elongating mitochondrial derivative of largely normal morphology. In Drp1 spermatids with connected Nebenkerne, one would expect normal unfurling and elongation to lead to a maximum of eight distinct linear mitochondrial derivatives, perhaps still connected. In contrast, the mitochondrial material in Drp1 spermatids spreads out, appears massively interconnected and tangled, and does not elongate. It is therefore speculated that the lack of mitochondrial fission in Drp1 spermatids may directly interfere with mitochondrial unfurling, thereby inhibiting mitochondrial elongation (Aldridge, 2007).

In neurons, both Drp1 and Milton are required for proper transport of mitochondria to synapses. However, the basis for the defects appears to be different in each case; in Drp1 homozygous neurons, a presumed failure of mitochondrial division leads indirectly to the unavailability of small transportable mitochondria, while in milton neurons, the defect appears to be more directly in the transport process. It was found that in spermatogenesis, the Drp1 and milton mutant phenotypes are distinct, confirming separate roles for these genes in mitochondrial morphogenesis (Aldridge, 2007).

The data are consistent with a role for Milton in some events of mitochondrial distribution in Drosophila spermatids. In spermatids derived from homozygous milt₉₂ spermatocytes, Nebenkerne form properly, indicating that Milton is not required for mitochondrial aggregation. However, the Nebenkerne remain beside the nucleus only 36% of the time, compared to 89% in wild type spermatids. Milton thus contributes to proper anchoring of the Nebenkern in onion stage spermatids, though other gene products also must play a role in this attachment. In wild type cells at this stage, the Nebenkern resides directly beside the spot where the basal body is embedded in the nucleus. The axonemal microtubules emanating from the basal body are surrounded by a membraneous sheath, and the Nebenkern associates not with the axonemal microtubules directly but instead with cytoplasmic microtubules that also emanate from the basal body. Perhaps Milton, via an association with kinesin, helps connect the Nebenkern in stable fashion to cytoplasmic microtubules anchored to the nucleus (Aldridge, 2007).

Milton also plays a role in the elongation of mitochondria during axonemal growth. In wild type cells, when the two mitochondrial derivatives within a Nebenkern unfurl from each other, they very transiently appear as two round lobes (and are very rarely observed at this stage) but then are immediately distorted, pulled lengthwise into a leaf blade shape, presumably along the cytoplasmic microtubules. In spermatids derived from homozygous milt₉₂ spermatocytes, the unfurled mitochondrial derivatives appear as spherical lobes for an extended period of time, suggesting that Milton normally mediates attachment of mitochondria to the cytoplasmic microtubules to enable shape changes. In wild type cells of a slightly later stage, the increasingly available surface area from the unfurling mitochondrial derivatives allows further elongation of the leaf blade structure. In contrast, the unfurling mitochondrial derivatives in milt₉₂ spermatids are not immediately stretched along the cytoplasmic microtubules, remaining crumpled. This early failure of elongation occurs whether or not the mitochondrial derivatives have maintained association with the nucleus. Ultimately, some mitochondrial elongation occurs in milt₉₂ spermatids, though mitochondrial derivatives in these cells are misshapen and usually oriented improperly with respect to the nucleus. It is concluded that Milton plays an important role in mitochondrial elongation, likely through attachment to microtubules, but that other gene products mediate some mitochondrial elongation in the absence of Milton (Aldridge, 2007).

The elongation of spermatid mitochondria may involve either (1) mitochondrial anchoring at the proximal (minus) end of cytoplasmic microtubules and subsequent sliding of mitochondrial membranes toward the distal (plus) end, or (2) progressive immobilization of mitochondrial membranes along growing cytoplasmic microtubules, analogous to the closing of a zipper. Milton (and perhaps kinesin) may act as they do in neurons, mediating mitochondrial movement toward the microtubule plus ends, or may serve simply to anchor mitochondria in static fashion during elongation. The decreased association of Nebenkerne with nuclei in milt₉₂ onion stage spermatids also suggests an anchoring role for Milton at the microtubule minus end. The dynein motor protein has recently been shown to act not only as a progressive motor but also as a static anchor for cargo. Consistent with a bidirectional transport model, the non-kinesin-associated Milton isoform (Glater, 2006) and/or the testis-specific isoform (Stowers, 2002) may enable anchoring or minus-end directed movement of mitochondria toward the nucleus, while other isoforms may mediate plus.end directed mitochondrial elongation (Aldridge, 2007).

The technique udrf for generating germline clones of Drp1 and milton allowed assessment of mutant phenotypes through the mid-elongation stages of spermatogenesis, after which point the condensed nuclei and the bundled nature of the elongating sperm (sixty four cells per cyst) made it impossible to identify individual mutant cells within a cyst. The transheterozygous male flies did not have homozygous clones that encompassed entire cysts. Most structural defects during spermatogenesis cause sterility through failure of individualization, which is the final investment of each sperm with its own plasma membrane and concomitant disposal of waste materials from each cell. Given the severity of the Drp1 and milton phenotypes, it is likely that individualization of mutant sperm similarly fails in these mutants. Observations that homozygous milton germline clones in a heterozygous dominant male sterile background do not confer fertility, while clones of the background chromosome do, indeed suggest that milton sperm either fail to individualize or are non-motile due to the mitochondrial defects (Aldridge, 2007).

In summary, roles have been defined for Drp1 and Milton in the specialized mitochondrial morphogenesis that takes place during spermatogenesis in Drosophila. Drp1-mediated mitochondrial division enables proper mitochondrial distribution during male meiosis as well as post-meiotic unfurling of mitochondrial derivatives (either directly or indirectly). Milton helps anchor the Nebenkern to the nucleus and subsequently mediates elongation of mitochondrial derivatives in developing spermatids. This work demonstrates that similar mechanisms for mitochondrial morphogenesis have been adapted for highly specialized use in different tissues within the organism (Aldridge, 2007).

Synaptic mitochondria are critical for mobilization of reserve pool vesicles at Drosophila neuromuscular junctions

In a forward screen for genes affecting neurotransmission in Drosophila, mutations were identified in dynamin-related protein (drp1). DRP1 is required for proper cellular distribution of mitochondria, and in mutant neurons, mitochondria are largely absent from synapses, thus providing a genetic tool to assess the role of mitochondria at synapses. Although resting Ca2+ is elevated at drp1 NMJs, basal synaptic properties are barely affected. However, during intense stimulation, mutants fail to maintain normal neurotransmission. Surprisingly, FM1-43 labeling indicates normal exo- and endocytosis, but a specific inability to mobilize reserve pool vesicles, which is partially rescued by exogenous ATP. Using a variety of drugs, evidence is provided that reserve pool recruitment depends on mitochondrial ATP production downstream of PKA signaling and that mitochondrial ATP limits myosin-propelled mobilization of reserve pool vesicles. These data suggest a specific role for mitochondria in regulating synaptic strength (Verstreken, 2005).

DRP1 is the fly homolog of dynamin-related protein, a protein implicated in fission of the outer mitochondrial membrane (reviewed in Praefcke, 2004 and Rube, 2004). Mutations in Drosophila drp1 lead to dramatic defects in synaptic localization of mitochondria, but not in that of other organelles. The animals survive beyond the third instar larval stage and sometimes develop into adult flies. These and other data indicate that mitochondria in the cell bodies of the mutants are still functional. Hence, drp1 mutants allow assessment of the role of mitochondria in neurotransmission. The data show that when drp1 synapses are stimulated at high frequency, they fail to maintain normal neurotransmission. Interestingly, this defect is not due to defects in exo- or endocytosis. Rather, the data indicate that lack of synaptic mitochondria results in a specific defect in mobilizing reserve pool (RP) vesicles. Furthermore, the addition of ATP partially rescues the observed defects. Similarly, application of drugs that target mitochondrial ATP production, but not Ca2+ buffering, block RP mobilization, suggesting that mitochondrial energy production is critical for RP mobilization. Using inhibitors of myosin light chain kinase, evidence is provided that ATP production by mitochondria is limiting to myosin-propelled vesicle mobilization from the RP, downstream of PKA-mediated mobilization of RP vesicles. Hence, these data suggest a regulatory role for mitochondria in the control of synaptic strength (Verstreken, 2005).

Most studies that acutely perturb mitochondria indicate that these organelles rapidly (<5 s) buffer Ca2+ during intense stimulation. However, in drp1, in which mitochondria are largely lacking at the synapse (this work; Li, 2004), Ca2+ elevation after 30 s of 10 Hz stimulation is similar to that in controls, suggesting that mitochondria play no or a minor role in Ca2+ buffering at NMJs under the conditions tested. Instead, other Ca2+ clearance mechanisms, such as the ER or the Na+/Ca2+ pumps, may predominate early during stimulation. Interestingly, when Ca2+ uptake into the ER is blocked at the Drosophila NMJ, intense stimulation induces a steep rise in intracellular Ca2+ that quickly returns to control levels, suggesting that the ER functions as an immediate Ca2+ sink. In addition, recruitment of mitochondrial Ca2+ buffering only after prolonged stimulation supports the existence of low-affinity Ca2+. Hence, this analyses provide evidence that mitochondria are not the main determinant of Ca2+ regulation during intense stimulation at the Drosophila NMJ (Verstreken, 2005).

When mitochondrial function is acutely blocked during intense stimulation, synaptic transmission is depressed. Ultrastructural analyses in amphibian synapses have attributed this to a reduced total number of vesicles, whereas FM1-43 studies in snake terminals and sesB mutants in flies suggest impairments in vesicle cycling. Although these studies imply a function for mitochondria in neurotransmission, the role of these organelles in regulating vesicle cycling has remained elusive. These data indicate that mitochondria are central to RP vesicle cycling. (1) Neurotransmission in drp1 mutants attenuates during intense stimulation. (2) Specific FM loading protocols mostly fail to label the RP of drp1 mutants. (3) RP loading is disrupted in NMJs treated with drugs that poison mitochondrial ATP production, but not in those treated with a drug that impairs mitochondrial Ca2+ buffering. (4) Several of the functional defects described in this study are strikingly similar to those of NCAM-deficient mouse NMJs also harboring a defective reserve pool of vesicles or synapsin knockout mice lacking a reserve pool. Taken together, these observations indicate that the reserve pool vesicle cycling is disrupted in drp1 mutants (Verstreken, 2005).

A failure to label the reserve pool (RP) of drp1 mutants with FM1-43 could be caused by either a direct defect in loading or a defect in unloading of this vesicle pool, resulting in an inability to replace old unlabeled RP vesicles with new FM1-43-labeled vesicles. Although a defect in loading cannot be excluded, the data are consistent with defects in unloading and mobilization of the RP. First, when oligomycin, a blocker of mitochondrial ATP production, is added to preparations in which the RP was labeled with FM1-43, intense stimulation fails to unload the labeled RP vesicles, suggesting that mitochondrial ATP is required for their mobilization. Second, it is conceivable that a defect in RP loading results in a smaller total vesicle pool; however, the number of vesicles measured by TEM in drp1 and in controls at rest is similar. Finally, stimulation of shi;drp1 mutants at the restrictive temperature leads to the release of fewer vesicles than in controls, suggesting that drp1 mutants harbor nonreleasable RP vesicles. Interestingly, forward-filling of shi;drp1 motor neurons with ATP alleviates this defect. Hence, without excluding additional defects in the loading of RP vesicles, these data indicate that lack of synaptic ATP primarily affects the mobilization of RP vesicles (Verstreken, 2005).

It is generally assumed that mitochondria fuel many steps of the vesicle cycle, including NSF (N-ethylmaleimide-sensitive fusion protein)-mediated SNARE (soluble NSF attachment protein receptor) uncoupling and vesicle uncoating, transport, and priming, by providing ATP for these processes. However, this study provides evidence that mitochondria (when depleted by >90%) specifically limit RP mobilization and do not play a critical role in endo- or exocytosis. (1) During mild stimulation, EJPs and FM1-43 dye uptake are normal, indicating normal basal endo- and exo-cytosis. (2) After depleting the cycling vesicle pool of shi;drp1, these mutants are able to reform this pool in the same time frame as controls. (3) Although drp1 mutants exhibit rundown at intense stimulation, vesicle uptake into the ECP is normal during this paradigm. (4) drp1 boutons loaded with FM1-43 at the end of a 10 Hz train release these vesicles almost completely upon stimulation, indicating normal exocytosis. These observations demonstrate that defects in endo- and exocytosis do not account for the rundown observed during intense stimulation and suggest that synaptic mitochondrial function does not affect all steps of the vesicle cycle equally (Verstreken, 2005).

The data suggest that energy generation by mitochondria is critical for RP vesicle mobilization, as ATP partially rescues the functional defects in drp1 mutants. In addition, TPP+, a drug that affects mitochondrial Ca2+ buffering but not ATP production, shows normal mobilization of RP vesicles. In contrast, drugs that block mitochondrial ATP production show defects in RP mobilization. Finally, analyses in hippocampal synapses suggest that ECP cycling does not require much ATP, in agreement with the current observations (Verstreken, 2005).

The observation that a severe reduction in synaptic mitochondria rather specifically blocks RP mobilization suggests that this process requires most of the ATP during intense activity and that enough ATP persists in stimulated drp1 synapses to maintain exo- and endocytosis. Taking into account ATP synthesis by the few remaining mitochondria at drp1 synapses (5%-10%) and the ATP generated by glycolysis in the cytoplasm (5% of the total ATP production, it is estimated that drp1 synapses produce only 10%-15% of the ATP of control synapses. It is surmised that this is sufficient for exo-endo cycling vesicular pool cycling, but is limiting for RP mobilization (Verstreken, 2005).

The molecular mechanisms of vesicle mobilization remain, at present, poorly understood. However, an involvement of PKA and MLC has been inferred from several studies. Whereas PKA inactivation leads to increased RP vesicle tethering, active PKA results in their release. This study provides evidence that the mobilization of these untethered vesicles requires ATP produced by mitochondria, and the data further suggest that this ATP-dependent step involves myosin. The myosin complex organizes synaptic vesicle transport along actin tracts. Myosin is a major ATPase and is activated by MLCK-mediated phosphorylation of its light chain (MLC). These findings indicate that myosin uses mitochondrial-produced ATP to mobilize RP vesicles. Indeed, supplementing drp1 synapses with ATP rescues their RP mobilization defect. However, MLCK inhibitors block this effect, suggesting that activation of myosin by MLC and MLCK and the supply of mitochondrial ATP are required to mobilize RP vesicles. Recent data show that mitochondrial ATP production is regulated by synaptic activity, further highlighting the central importance of mitochondria in the regulation of synaptic strength and providing a direct link between synaptic activity and the mobilization of RP vesicles (Verstreken, 2005).

Mutations in dynamin-related protein result in gross changes in mitochondrial morphology and affect synaptic vesicle recycling at the Drosophila neuromuscular junction

Mitochondria are the primary source of ATP needed for the steps of the synaptic vesicle cycle. Dynamin-related protein (DRP) is involved in the fission of mitochondria and peroxisomes. To assess the role of mitochondria in synaptic function, a Drosophila DRP mutant combination was characterized that shows an acute temperature-sensitive paralysis. Sequencing of the mutant reveals a single amino acid change in the guanosine triphosphate hydrolysing domain (GTPase domain) of DRP. The synaptic mitochondria in these mutants are remarkably elongated, suggesting a role for DRP in mitochondrial fission in Drosophila. There is a loss of neuronal transmission at restrictive temperatures in electroretinogram (ERG) recordings. Like stress-sensitive B (sesB), a mitochondrial adenosine triphosphate (ATP) translocase mutant that was studied earlier for its effects on synaptic vesicle recycling, an allele-specific reduction in the temperature of paralysis of Drosophila synaptic vesicle recycling mutant shibire was seen in the DRP mutant background. These data, in addition to depletion of vesicles observed in electron microscopic sections of photoreceptor synapses at restrictive temperatures, suggest a block in synaptic vesicle recycling due to reduced mitochondrial function (Rikhy, 2007).

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Search PubMed for articles about Drp1 in all species

Abdelwahid, E., Yokokura, T., Krieser, R. J., Balasundaram, S., Fowle, W. H. and White, K. (2007). Mitochondrial disruption in Drosophila apoptosis. Dev. Cell 12(5): 793-806. PubMed citation: 17488629

Aldridge, A. C., Benson, L., Siegenthaler, M. M., Whigham, B. T., Stowers, R. S., Hales, K. G. (2007). Roles for Drp1, a dynamin-related protein, and milton, a kinesin-associated protein, in mitochondrial segregation, unfurling, and elongation during Drosophila spermatogenesis. Fly 1(1): 38-46. Article

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ClaverÌa. C., et al. (2002). GH3, a novel proapoptotic domain in Drosophila Grim, promotes a mitochondrial death pathway. EMBO J. 21: 3327-3336. PubMed citation: 12093734

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Rikhy, R., Kamat, S., Ramagiri, S., Sriram, V. and Krishnan, K. (2007). Mutations in dynamin-related protein result in gross changes in mitochondrial morphology and affect synaptic vesicle recycling at the Drosophila neuromuscular junction. Genes Brain Behavior 6: 42-53. PubMed citation: 17233640

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date revised: 5 March 2007

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