1.

Programmed cell death is a fundamental process during development

Programmed cell death appears as a very early event in the course of evolution allowing to limit the size of cellular populations and to eliminate some undesirable cells [START_REF] Ellis | Mechanisms and functions of cell death[END_REF]. This process is fundamental for the development of multicellular organisms, in the course of which many embryonic cells are brought to die. Programmed cell death, as does cell proliferation, intervenes in determination of the size and the form of organs, as well as in functional maturation of some systems.

During the development of members, cell proliferation and differentiation allow the appearance and the growth of member buds, while the morphogenesis of fingers and toes invokes the death of cells initially located in interdigital positions. During the development of the nervous system, neurons that do not reach their target are eliminated by a process of programmed cell death [START_REF] Thompson | Apoptosis in the pathogenesis and treatment of disease[END_REF].

This death allows to establish functions of the nervous system by playing on its plasticity. The functional maturation of the immune system also involves massive programmed cell death. The clones of self-reactive T lymphocytes are eliminated by a process of programmed cell death. In other cases, structures whose physiological role is only transitory are eliminated, for example during the metamorphosis of amphibians (tail of tadpoles) and insects (intersegmental muscles).

Apoptosis is a type of programmed cell death whose control is altered in several pathologies

What is the mechanism of programmed cell death? How is it controlled? Different types of programmed cell death can be defined, according to morphological criteria [START_REF] Clarke | Developmental cell death: morphological diversity and multiple mechanisms[END_REF][START_REF] Schwartz | Do all programmed cell deaths occur via apoptosis?[END_REF]. Apoptosis, defined in 1972 [START_REF] Kerr | Apoptosis: a basic biological phenomenon with wideranging implications in tissue kinetics[END_REF], is the most frequently described type of programmed cell death. Apoptosis is often opposed to necrosis, which is a cellular death that results generally from massive aggressions of the external medium. A dysfunction of apoptosis or of its control can be implicated in various pathologies [START_REF] Thompson | Apoptosis in the pathogenesis and treatment of disease[END_REF]. A defect in the cell death program could be responsible for some autoimmune diseases [START_REF] Tan | Autoimmunity and Apoptosis[END_REF]. It is also implicated in oncogenesis. Indeed, apoptosis plays a fundamental role in different steps of carcinogenesis [START_REF] Williams | Programmed cell death : apoptosis and oncogenesis[END_REF][START_REF] Bursch | Cell death by apoptosis and its protective role against disease[END_REF]. For example, in the course of hepatocarcinogenesis, initiated cells have an apoptotic activity that compensates the intense proliferative activity of the other cells. It results in elimination of most of the initiated cells. Apoptosis can also be observed at other stages of tumor progression as well as in other types of cancers. The control of cell proliferation by mitogens and apoptosis is always active in neoplasic cells and even in some malignant tumors with however an imbalance between the two phenomena. Some oncogens and oncosuppressor genes are now known for their action on apoptosis. The p53 oncosuppressor induces apoptosis in several transformed cell lines [START_REF] Yonish-Rouach | Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6[END_REF][START_REF] Shaw | Induction of apoptosis by wild-type p53 in a human colon tumor-derived cell line[END_REF] and the c-myc gene also seems to be involved in this process [START_REF] Evan | Induction of apoptosis in fibroblasts by c-myc protein[END_REF][START_REF] Shi | Role for c-myc in activation-induced apoptotic cell death in T cell hybridomas[END_REF]. On the contrary, the bcl-2 oncogene can block apoptosis in different situations and this capacity is probably involved in the genesis of lymphomas and many cancers [START_REF] Hockenbery | Bcl-2 is an inner mitochondrial protein that blocks programmed cell death[END_REF][START_REF] Sentman | Bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes[END_REF][START_REF] Strasser | Bcl-2 transgene inhibits T-cell death and perturbs thymic self-censorship[END_REF].

Others pathologies can be due on the contrary to an ectopic induction of apoptosis. Some neuro-degenerative diseases, during which a massive neuronal death is observed, could involve apoptosis. This could be the case for Alzheimer and Parkinson diseases and for the amyotrophic lateral sclerosis [START_REF] Thompson | Apoptosis in the pathogenesis and treatment of disease[END_REF]. This same phenomenon could also be involved in the pathogenesis of AIDS where it would lead to the elimination of T lymphocytes and then to immunodeficiency [START_REF] Gougeon | Apoptosis in AIDS[END_REF][START_REF] Martin | Programmed cell death and AIDS[END_REF][START_REF] Ameisen | The relevance of apoptosis to AIDS pathogenesis[END_REF].

Programmed cell death is genetically controlled

The genetic approach, with the nematode Caenorhabditis elegans, has allowed to show that condemned cells express genes that participate actively in apoptosis (ced-3, ced-4). If the coordinated expression of these genes is disturbed, cells do not die. A gene, ced-9, capable of blocking apoptosis has also been found. Ced-9 is the homologue of bcl-2 that belongs to a family of genes that inhibits or stimulates apoptosis. Ced-3 is the homologue of a family of genes of cystein proteases (caspases) to which belongs the gene ICE that codes for the enzyme of conversion of interleukin-1 (table 1). More recently an homologue of Ced-4, called Apaf-1 has also been identified [START_REF] Zou | Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3[END_REF]. These observations underline that this process appears early during evolution. The mode of action of genes of the bcl-2 family is poorly understood and the substrates of the caspases really implicated in apoptosis remain to be ascertained [START_REF] Kumar | ICE-like proteases in apoptosis[END_REF]Guénal et al., 1997a). In mammals, many other genes have been described as having a negative or positive action on apoptosis. Some of these genes are known to be involved in the control of cellular proliferation, this being in agreement with the idea that death by apoptosis allows to compensate cellular multiplication and that it is involved in protection against cancers. Furthermore, many viral gene products are inhibitors of apoptosis that act as antagonists of cellular proteins implicated in apoptosis (table 2). This observation shows that these different viruses have developed, in the course of evolution, different strategies to avoid the death of the infected cell before the end of the viral cycle [START_REF] Gillet | Viral inhibition of apoptosis[END_REF]. Then, cells are committed to the degradation phase where they present morphological (condensation of the cell, the nucleus and the chromatin) and molecular (fragmentation of the DNA in oligonucleosomal fragments) characteristics of apoptosis. In some cases, the phagocytosis of apoptotic cells occurs before these modifications become perceptible. Products of ced-3, ced-4 and ced-9 as well as of their mammalian homologues intervene during the effector phase. Several proteins involved in the phase of degradation (ubiquitin, transglutaminase, nuclease) have also been identified.

The different ways of induction of apoptosis

5.

A drop of the mitochondrial membrane potential is a universal and early event of apoptosis What is the place of mitochondria in apoptosis? The mitochondrial membrane potential ( m ) can be estimated with fluorochromes that incorporate into mitochondria as a function of their potential, according to the Nernst equation (table 3). The commitment to apoptosis is accompanied by a drop of the  m that occurs before the fragmentation of DNA in oligonucleosomal fragments. At least in the case of apoptosis of fibroblasts immortalized by a temperature sensitive mutant of SV40

LT antigen, where apoptosis is mediated by the p53 protein [START_REF] Zheng | Apoptosis is antagonized by Large T antigens in the pathway to immortalization by polyomaviruses[END_REF], this drop of  m is responsible for a defect of maturation of mitochondrial proteins synthesized in the cytoplasm [START_REF] Mignotte | SV40 induced cellular immortalization : phenotypic changes associated with the loss of proliferative capacity in a conditionally immortalized cell line[END_REF], cessation of mitochondrial translation and an uncoupling of the oxidative phosphorylations [START_REF] Vayssière | Commitment to apoptosis is associated with changes in mitochondrial biogenesis and activity in cell lines conditionally immortalized with Simian Virus 40[END_REF]. The drop of  m is detectable whatever the apoptotic induction signal, physiological (absence of growth factor, glucocorticoids, TNF) or non-physiological (irradiation, chemotherapy) [START_REF] Petit | Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocytes apoptosis[END_REF]Zamzami et al., 1995a;Zamzami et al., 1995b;[START_REF] Marchetti | Apoptosis-associated derangement of mitochondrial function in cells lacking mitochondrial DNA[END_REF][START_REF] Sidoti-De Fraisse | TNF- activates at least two apoptotic signaling cascades[END_REF]. Therefore, it seems that the drop of  m , an event that can be slowed down by cyclosporine A, is a universal characteristic that accompanies apoptosis, independently of the induction signal and of the cellular type [START_REF] Kroemer | The biochemistry of programmed cell death[END_REF]. These data show on the one hand that the nuclear fragmentation is a late event as compared to the drop of the  m and, on the other hand, that this drop marks the point of no-return of a cell condemned to die.

The measure of  m allows identification of cells in "pre-apoptosis", including in circulating T lymphocytes from human immunodeficiency virus-1 (HIV-1) carriers [START_REF] Macho | Mitochondrial dysfunctions in circulating T lymphocytes from human immunodeficiency virus-1 carriers[END_REF].

A kinetic analysis of splenocyte, T cell hybridoma and pre-B cell apoptosis realized with the help of hydroethidine (table 3) has permitted to show a reactive oxygen species (ROS) production consecutive to the drop of  m (Zamzami et al., 1995a). This step can be selectively inhibited by rotenone (inhibitor of the complex I of the respiratory chain) and by ruthenium red (inhibitor of the uniport of calcium of the inner mitochondrial membrane). The increased production of ROS seems to be accompanied by local effects on the cardiolipins of the inner mitochondrial membrane. The alteration of the cardiolipins is detectable by labeling cells with the probe 10-nonyl-acridine orange (table 3) that binds in a stoechiometric manner to the intact cardiolipins of the inner mitochondrial membrane. At this stage, cells possess a normal size and a plasma membrane whose permeability is not altered. These changes are visible only in an ulterior step coincident with a diminution of the cell size, an increase of membrane permeability and also degradation of the nuclear DNA [START_REF] Kroemer | The biochemistry of programmed cell death[END_REF]. This last step can be delayed by compounds that eliminate free radicals, which suggests the implication of ROS in the execution of the apoptotic process.

Direct interventions on mitochondrial permeability transition modulate apoptosis

The permeability transition (PT), phenomenon inhibited by cyclosporine A, is characterized by the opening of mitochondrial megachannels that allows permeability to compounds of molecular weight < 1500 Da, a reduction of the  m and the arrest of ATP synthesis [START_REF] Bernardi | Modulation of the mitochondrial permeability transition pore. Effect of protons and divalent cations[END_REF][START_REF] Petronilli | Regulation of the permeability transition pore, a voltage-dependent mitochondrial channel inhibited by cyclosporin A[END_REF]. Interestingly, permeability transition has properties of self-amplification.

Indeed, the drop of the  m , that is linked to depletion of non-oxidized glutathione [START_REF] Macho | Glutathione depletion is an early and calcium elevation is a late event of thymocyte apoptosis[END_REF] and that result from the opening of the PT pores would increase the permeability transition in a retrograde manner [START_REF] Ichas | Mitochondria are excitable organelles capable of generating and conveying electrical and calcium signals[END_REF]. We have therefore proposed that the opening of the PT pore may constitute an irreversible state of the effector phase of apoptosis and could account for the apparent synchronization in the drop of  m that takes place simultaneously in all the mitochondria of a same cell [START_REF] Kroemer | The biochemistry of programmed cell death[END_REF]. The molecular composition of these megachannels is not entirely known. The peripheral benzodiazepin receptor, that has recently been implicated in the protection against ROS [START_REF] Carayon | Involvement of peripheral benzodiazepine receptors in the protection of hematopoietic cells against oxygen radical damage[END_REF], and the translocase of adenine nucleotides (ANT) are components of this channel. Indeed, among compounds described as being able to regulate the PT are two specific inhibitors to the ANT: atractyloside and bongkrekic acid (BA). These two molecules could favor different conformations of the ANT: a closed conformation induced by BA and

cyclosporine and an open conformation induced by atractyloside. The drop of  m induced by the protoporphyrine IX, a ligand specific of the peripheral benzodiazepin receptor (that binds to ANT), entails apoptosis (Zamzami et al., 1996a). On the contrary, N-methyl Val-4-cyclosporin A (a derivative of cyclosporine that is not immunosuppressor) and bongkrekic acid, prevent the drop of the mitochondrial potential and the consecutive fragmentation of the DNA. These data show that the induction of PT provokes apoptosis while its inhibition provides a protection against it. These results sustain the hypothesis that permeability transition is involved in the drop of  m observed during the effector phase of apoptosis [START_REF] Kroemer | The biochemistry of programmed cell death[END_REF][START_REF] Petit | Mitochondria and programmed cell death: back to the future[END_REF][START_REF] Mignotte | Mitochondria and apoptosis[END_REF].

Mitochondria can control nuclear apoptosis

Direct alterations of mitochondria can induce apoptosis [START_REF] Hartley | Complex I inhibitors induce dosedependent apoptosis in PC12 cells: relevance to Parkinson's disease[END_REF][START_REF] Wolvetang | Mitochondrial respiratory chain inhibitors induce apoptosis[END_REF]. The links between mitochondrial perturbations and nuclear alterations can be studied by means of an acellular system where purified nuclei and purified mitochondria are confronted [START_REF] Newmeyer | Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria[END_REF]. Such a system allows on the one hand to study reciprocal and direct effects of one organelle on another and, on the other hand, to characterize at the biochemical level the factors involved. The result of such a manipulation has shown that normal mitochondria have no effect on isolated cell nuclei, while the processing of mitochondria with compounds capable to induce the permeability transition (PT) is sufficient to provoke nuclear apoptosis (condensation of the chromatin and fragmentation of the DNA) (Zamzami et al., 1996b). A strict correlation between induction of the PT and nuclear apoptosis has been observed by using a variety of known inductors of the PT such as atractyloside, pro-oxidants, calcium, protonophores and substances that provoke linkage of thiol groups such as diamide. These compounds, that have no direct effect on nuclei in absence of mitochondria, confer pro-apoptotic properties upon mitochondria. The pro-apoptotic character (induction of nuclear apoptosis) of the mitochondria treated with atractyloside is altered by inhibitors of the PT such as bongkrekic acid, cyclosporine A and compounds like monochlorobimane that block the cross-linking of the thiols. Cyclosporine A can be replaced by its non immunosuppressor analogue, N-methyl Val-4-cyclosporine A, which shows that its inhibitory effect on PT and nuclear apoptosis is independent of its calcineurine activity. These results suggest the implication of the PT in the regulation of apoptosis induced via the mitochondria.

Finally, mitochondria isolated from apoptotic cells in vivo are capable of inducing nuclear apoptosis in the acellular system. Indeed, the induction of mouse hepatocyte apoptosis in vivo by a combination of D-galactosamine and lipopolysaccharide entails the reduction of  m . The mitochondria isolated from these cells provoke apoptosis of HeLa cell nuclei in vitro. A similar result has been obtained with mitochondria isolated from cells of spleen processed with dexamethasone.

These results show that mitochondria can effectively control nuclear apoptosis (Zamzami et al., 1996b). The mitochondria that undergo the PT would liberate a protein capable of inducing nuclear apoptosis. Some works suggest that the cytochrome c, by dissociating from the mitochondria, activate proteases implicated in nuclear apoptosis [START_REF] Liu | Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c[END_REF][START_REF] Kluck | The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis[END_REF][START_REF] Yang | Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked[END_REF].

Other results suggest that the "apoptogenic" protein derived from the mitochondria is a protease of approximately 50 kDa, whose activity is sensitive to the tri-peptide Z-VAD.fmk (a caspase inhibitor) [START_REF] Susin | Bcl-2 inhibits the mitochondrial release of an apoptogenic protease[END_REF].

Bcl-2 inhibits permeability transition and the release of cytochrome c and AIF in the cytoplasm

Data concerning Bcl-2 underline the role of mitochondria in apoptosis [START_REF] Kroemer | The proto-oncogene Bcl-2 and its role in regulating apoptosis[END_REF][START_REF] Reed | Double identity for proteins of the Bcl-2 family[END_REF][START_REF] Mignotte | Mitochondria and apoptosis[END_REF]. Bcl-2, and other related proteins, are localized in intracellular membranes and specially in the external membrane of mitochondria [START_REF] Krajewski | Investigation of the subcellular distribution of the bcl-2 oncoprotein: residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes[END_REF][START_REF] Nakai | The bcl-2 protein is inserted into the outer membrane but not into the inner membrane of rat liver mitochondria in vitro[END_REF][START_REF] Nguyen | Targeting of bcl-2 to the mitochondrial outer membrane by a COOH-Terminal signal anchor sequence[END_REF][START_REF] Akao | Multiple subcellular localization of Bcl-2: detection in nuclear outer membrane, endoplasmic reticulum membrane, and mitochondrial membranes[END_REF][START_REF] De Jong | Subcellular localization of the bcl-2 protein in malignant and normal lymphoid cells[END_REF][START_REF] Gonzalez-Garcia | bcl-X L is the major bcl-x mRNA form expressed during murine development and its product localizes to mitochondria[END_REF][START_REF] Hickish | Ultrastructural localization of BHRF1: an Epstein-Barr virus gene product which has homology with bcl-2[END_REF][START_REF] Janiak | Assembly of Bcl-2 into microsomal and outer mitochondrial membranes[END_REF][START_REF] Yang | The intracellular distribution and pattern of expression of Mcl-1 overlap with, but are not identical to, those of Bcl-2[END_REF]. Anti-apoptotic properties of Bcl-2 are affected in absence of its segment of anchorage to membranes [START_REF] Tanaka | Structure-function analysis of the Bcl-2 oncoprotein. Addition of a heterologous transmembrane domain to portions of the Bcl-2 beta protein restores function as a regulator of cell survival[END_REF][START_REF] Nguyen | Role of membrane anchor domain of Bcl-2 in suppression of apoptosis caused by E1B-defective adenovirus[END_REF][START_REF] Zhu | Bcl-2 mutants with restricted subcellular location reveal spatially distinct pathways for apoptosis in different cell types[END_REF]. However, it has been shown that bcl-2 can block apoptosis of cells devoid of mitochondrial DNA ( 0 cells) [START_REF] Jacobson | Bcl-2 blocks apoptosis in cells lacking mitochondrial DNA[END_REF]. These experiments show that, in these cells that have undergone a complex selection and that possess mitochondria that have a  m close to the normal [START_REF] Skowronek | A fluorescence-microscopic and flow cytometric study of HeLa cells with an experimentally induced respiratory deficiency[END_REF][START_REF] Marchetti | Apoptosis-associated derangement of mitochondrial function in cells lacking mitochondrial DNA[END_REF][START_REF] Sidoti-De Fraisse | TNF- activates at least two apoptotic signaling cascades[END_REF], the antiapoptotic activity of bcl-2 can always be exerted. However, they exclude neither that the antiapoptotic effect of bcl-2 is linked to an activity that is always present in mitochondria of  0 cells nor that its anti-apoptotic power exerts at several levels. It has effectively been shown that Bcl-2, in contrast to inhibitors of caspases, blocks the drop of  m that occurs during the cellular death induced by respiratory inhibitors [START_REF] Shimizu | Bcl-2 blocks loss of mitochondrial membrane potential while ICE inhibitors act at a different step during inhibition of death induced by respiratory chain inhibitors[END_REF]. The importance of the structural location of Bcl-2 in nuclei or mitochondria as compared to its anti-apoptotic function has been studied. These organelles have been purified from hybridomas of T cells transfected by bcl-2 (Zamzami et al., 1996b). In acellular experiments, the treatment with atractyloside has shown that, in contrast to mitochondria purified from control cells, mitochondria purified from cells transfected by bcl-2 do not provoke nuclear apoptosis. On the contrary, nuclei purified from cells transfected by bcl-2 show a condensation of the chromatin and a fragmentation of the DNA when they are confronted to control mitochondria treated with atractyloside. Furthermore, bcl-2 inhibits the induction of permeability transition [START_REF] Decaudin | Bcl-2 and Bcl-X L antagonize the mitochondrial dysfunction preceding nuclear apoptosis induced by chemotherapeutic agents[END_REF] while bax promotes it [START_REF] Pastorino | The overexpression of bax produces cell death upon induction of the mitochondrial permeability transition[END_REF]. These results show that, even if Bcl-2 intervenes also during latter events (Guénal et al., 1997b), at least a part of its inhibitory activity on apoptosis is exerted by acting on the mitochondrial permeability transition.

Moreover, the structure of a protein of the Bcl-2 family (Bcl-x L ) has been recently established [START_REF] Muchmore | X-ray and NMR structure of human Bcl-X L , an inhibitor of programmed cell death[END_REF]. It recalls that of bacterial toxins, especially the diphtheria toxin, that form a pH-sensitive transmembrane channel. Furthermore, the pro-apoptotic Bax protein can form channels [START_REF] Antonsson | Inhibition of Bax Channel-Forming Activity by Bcl-2[END_REF], as reported also for the anti-apoptotic proteins Bcl-x L [START_REF] Minn | Bcl-x L forms an ion channel in synthetic lipid membranes[END_REF] and Bcl-2 [START_REF] Schendel | Channel formation by antiapoptotic protein Bcl-2[END_REF]. However, the intrinsic properties of Bax and those of Bcl-x L and Bcl-2 reveal differences. The channel forming activity of Bcl-x L and Bcl-2 is observed at highly acidic pH while Bax forms channel in a wide range of pH including at pH=7, that found in cells. Furthermore, Bcl-2 can block the pore-forming activity of Bax. These results strengthen the hypothesis that these proteins are components of the mitochondrial PT pores. Bax might promote cell death by allowing the efflux of ions and small molecules across the mitochondrial membranes, thus triggering permeability transition, while Bcl-2 might counteract this effect.

It has been shown that Bcl-2 inhibits the release of cytochrome c [START_REF] Kluck | The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis[END_REF][START_REF] Yang | Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked[END_REF] and of AIF [START_REF] Susin | Bcl-2 inhibits the mitochondrial release of an apoptogenic protease[END_REF] outside the mitochondria. Thus, one level of action of Bcl-2 is to control the efflux of proteins from the mitochondrial intermembrane space through the outer membrane and into the cytosol where caspases are found. In the same way, Bcl-x L inhibits AraCinduced preapoptotic accumulation of cytochrome c in the cytosol [START_REF] Kim | Overexpression of Bcl-X(L) inhibits Ara-C-induced mitochondrial loss of cytochrome c and other perturbations that activate the molecular cascade of apoptosis[END_REF] and cells that overexpress Bcl-x L fail to accumulate cytosolic cytochrome c or to undergo apoptosis in response to genotoxic stress. These findings support the hypothesis that Bcl-x L , as well as Bcl-2, protect cells from apoptosis by inhibiting the availability of cytochrome c in the cytosol. The mechanism allowing for the release of AIF and cytochrome c needs further investigations. PT pores, which are thought to connect the mitochondrial matrix to the cytosol, within the contact sites between inner and outer mitochondrial membranes [START_REF] Beutner | Complexes between kinases, mitochondrial porin and adenylate translocator in rat brain resemble the permeability transition pore[END_REF] are only permeable to small compounds (molecular mass <1500 Da). Thus PT pores are probably not the structure directly responsible for the efflux of these intermembrane space apoptogenic proteins. Future works will have to determine the way used by AIF and cytochrome c to leave the mitochondria. Opening of the PT pore could be just a first step of a cascade of events causing an increase in the permeability of the outer mitochondrial membrane.

Some results suggest that this permeability increase is due to mechanistic disruption secondary to an increase in matrix volume [START_REF] Scarlett | Release of apoptogenic proteins from the mitochondrial intermembrane space during the mitochondrial permeability transition[END_REF][START_REF] Van Der Heiden | Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria[END_REF].

Bcl-2 family proteins relocalize cellular proteins to mitochondrial membranes

Bcl-2 has also been found to interact with several other cellular proteins that do not belong to the Bcl-2 related proteins family. These proteins include Nip1, Nip2, and Nip3, the function of which is unknown [START_REF] Boyd | Adenovirus E1B 19 kDa and Bcl-2 proteins interact with a common set of cellular proteins[END_REF], the GTPase R-ras p23 [START_REF] Fernandez | Bcl-2 associates with the ras-related protein R-ras p23[END_REF], Raf-1 [START_REF] Wang | Apoptosis regulation by interaction of Bcl-2 protein and Raf-1 kinase[END_REF][START_REF] Ali | The study of cell death proteins in the outer mitochondrial membrane chemical cross-linking[END_REF], BAG-1 [START_REF] Takayama | Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity[END_REF], the cellular prion protein (PrP) [START_REF] Kurschner | The cellular prion protein (PrP) selectively binds to Bcl-2 in the yeast two-hybrid system[END_REF], the p53 binding-protein p53-BP2 [START_REF] Naumovski | The p53-binding protein 53BP2 also interacts with Bcl-2 and impedes cell cycle progression at G2/M[END_REF], the protein phosphatase calcineurin [START_REF] Shibasaki | Suppression of signalling through transcription factor NF-AT by interactions between calcineurin and Bcl-2[END_REF] and the mitochondrial membrane protein carnitine palmitoyltransferase I [START_REF] Paumen | Direct interaction of the mitochondrial membrane protein carnitine palmitoyltransferase I with Bcl-2[END_REF]. At least some of these interactions could reflect the ability of Bcl-2 to relocalize cellular proteins to mitochondrial membranes.

Indeed, a connection has been made between members of the CED-9/Bcl-2 family and caspases. Mutations that reduce or eliminate CED-9 activity also disrupt its ability to bind CED-4 [START_REF] Spector | Interaction between the C. elegans cell-death regulators CED-9 and CED-4[END_REF]. CED-9 (and Bcl-x L ) was found to interact with and inhibit the function of CED-4. Furthermore, CED-4 can simultaneously interact with CED-3 [START_REF] Irmler | Direct physical interaction between the Caenorhabditis elegans 'death proteins' CED-3 and CED-4[END_REF] (or mammalian caspases (Chinnaiyan et al., 1997b)). Thus, CED-9 might control C. elegans cell death by binding to and regulating CED-4 and CED-3 activities. A similar mechanism could also exist in mammalian cells. Bcl-x L interacts with caspase-1 (Chinnaiyan et al., 1997a) and inhibits the function of CED-4 (Chinnaiyan et al., 1997b). Furthermore, the human protease activating factor (Apaf-1), which participates in the cytochrome c-dependent activation of caspase-3, contains a caspaserecruitment domain (CARD). Altogether, these results suggest that Apaf-1, like the nematode CED-4, acts by activating caspases and could provide the physical link between Bcl-2 related proteins and caspases. [START_REF] Zou | Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3[END_REF]. Since Bax and Bik can disrupt the association between CED-9 (or Bcl-xL) and CED-4 (Chinnaiyan et al., 1997b), it is tempting to speculate that Bcl-x L and possibly other members of the Bcl-2 protein family inhibit apoptosis by maintaining the procaspases/Apaf-1 complexes associated to mitochondrial membranes and that Bax and Bik by dissociating the complexes permit the activation of procaspases.

Mitochondria participates in apoptosis signaling

Recently, it has appeared that mitochondria , in addition to their role during the effector phase of apoptosis, also intervene, in some cases, during the initiation phase of apoptosis. Much of the available data converge to the hypothesis that when ROS are involved in apoptosis signaling, they are the consequence of an impairment of the mitochondrial respiratory chain [START_REF] Schulze-Osthoff | Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial -21 -functions. Evidence for the involvement of mitochondrial radical generation[END_REF][START_REF] France-Lanord | Mitochondrial free radical signal in ceramide-dependent apoptosis: a putative mechanism for neuronal death in Parkinson's disease[END_REF][START_REF] Gudz | Direct inhibition of mitochondrial respiratory cahin complex III by cell-permeable ceramide[END_REF][START_REF] Quillet-Mary | Implication of mitochondrial hydrogen peroxide generation in ceramide-induced apoptosis[END_REF][START_REF] Sidoti-De Fraisse | TNF- activates at least two apoptotic signaling cascades[END_REF]. What is the mechanism leading to the accumulation of mitochondrial ROS? Much of the available data converge to the hypothesis that ROS increases are the consequence of an impairment of the mitochondrial respiratory chain [START_REF] Schulze-Osthoff | Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial -21 -functions. Evidence for the involvement of mitochondrial radical generation[END_REF][START_REF] France-Lanord | Mitochondrial free radical signal in ceramide-dependent apoptosis: a putative mechanism for neuronal death in Parkinson's disease[END_REF][START_REF] Gudz | Direct inhibition of mitochondrial respiratory cahin complex III by cell-permeable ceramide[END_REF][START_REF] Quillet-Mary | Implication of mitochondrial hydrogen peroxide generation in ceramide-induced apoptosis[END_REF]. Indeed, it was shown that an upstream inhibition, with chemical compounds acting on complex I (Schulze- [START_REF] Schulze-Osthoff | Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial -21 -functions. Evidence for the involvement of mitochondrial radical generation[END_REF][START_REF] Quillet-Mary | Implication of mitochondrial hydrogen peroxide generation in ceramide-induced apoptosis[END_REF], or an elimination of the electron transfer chain by depletion of the mtDNA prevent ROS accumulation and consequently protect cells against apoptosis induced by ceramide [START_REF] Quillet-Mary | Implication of mitochondrial hydrogen peroxide generation in ceramide-induced apoptosis[END_REF] or TNF- [START_REF] Sidoti-De Fraisse | TNF- activates at least two apoptotic signaling cascades[END_REF]. The ubiquinone site in complex III appears as the major site of mitochondrial ROS production as this site catalyzes the conversion of molecular oxygen to superoxide anion which can lead to the formation of other potent ROS such as hydrogen peroxide and hydrogen radicals. Such a model is supported by the observed potentiation of cell death processes in ROS-dependent apoptosis when electron flow was inhibited distal to the ubiquinone pool. However, the origin of these electron flow disturbances is not clear. The recently observed efflux of cytochrome c from mitochondria to cytosol in the early phases of apoptosis could provide a clue to resolve this question [START_REF] Liu | Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c[END_REF][START_REF] Kluck | The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis[END_REF][START_REF] Yang | Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked[END_REF]. Indeed, the release of cytochrome c must lead to a breakdown of the mitochondrial electron flow downstream of the ubiquinone site which in turn would result in an increased generation of ROS. Such a model is supported by the described correlation between loss of cytochrome c and respiratory failure in a Fasinduced apoptosis model [START_REF] Krippner | Loss of Function of Cytochrome c in Jurkat Cells Undergoing Fas-mediated Apoptosis[END_REF].

 (Sidoti

Conclusions

In conclusion, mitochondria are involved in the decision of cells to survive or not at several levels (figure 2). At a first level, mitochondria can contribute to apoptosis signaling, as shown in Second, after the induction phase, mitochondria participates in the activation of executing caspases responsible for the morphological changes in two ways. On the one hand, Ced-4/Ced-3 (Apaf-1/caspase) complexes dissociate from intracellular membranes and are released in the cytosol.

Legende to figures

On the other hand, the opening of PT pores is a first step of a cascade of events causing an increase in the permeability of the outer mitochondrial membrane, an efflux of proteins as AIF or cytochrome c, an increase in ROS production and an activation of executing caspases.

-25 - Pro-apoptotic: Bax, Bak, Bcl-x S , Bad, Bik, Hrk…

C. elegans Mammals

CED

Table 1: Programmed cell death in nematodes and mammals is controled by homologous genes.

Mammalian caspases act either during the activation or the execution phase of PCD. In mammals some members of the Bcl-2 related proteins are death antagonists while other are death agonists.

-26 - Table 3: Some fluorochromes used for the study of mitochondrial and nuclear alterations during apoptosis.

Pro-apoptotic cellular proteins

-28 -

seem to converge to a common effector phase Figure 1

 phase1 Figure1presents a simplified model of events occurring during apoptosis. Three phases can

Figure 1 :

 1 Figure 1:Simplified model of events occurring during apoptosis.

Figure 2 :

 2 Figure 2:Roles of mitochondria during apoptosis.

  Anti-apoptotic: Bcl-2, Bcl-x L , Bcl-w, Bfl-1, Brag-1, Mcl-1, A1, NR13…

  many viral genes products act as antagonists of pro-apoptotic cellular proteins. breaks by the action of DNA polymerase or terminal desoxyribonucleotidyl transferase.

  Figure 1

Figure 2

 2 Figure 2

TNF--or ceramide-induced cell death during which increased mitochondrial ROS production appears as an early event of the induction phase. At a second level, mitochondria are involved in the control of the activation of the cell death machinery by docking at their surface, via Bcl-2 family proteins, execution caspases or by sequestering, in the intermembrane space, caspase activators as AIF or cytochrome c.


Tommi Koskinen

Adam P Showman

M. S Marley

Cuzzi, J. N Kitzmann

Ricci Kempton

Typeset using L A T E X twocolumn style in AASTeX62

Keywords:

We study the properties of photochemical hazes in super-Earths/mini-Neptunes atmospheres with particular focus on GJ1214b. We evaluate photochemical haze properties at different metallicities between solar and 10000×solar. Within the four orders of magnitude change in metallicity, we find that the haze precursor mass fluxes change only by a factor of ∼3. This small diversity occurs with a non-monotonic manner among the different metallicity cases, reflecting the interaction of the main atmospheric gases with the radiation field. Comparison with relative haze yields at different metallicities from laboratory experiments reveals a qualitative similarity with our theoretical calculations and highlights the contributions of different gas precursors. Our haze simulations demonstrate that higher metallicity results into smaller average particle sizes. Metallicities at and above 100×solar with haze formation yields of ∼10% provide enough haze opacity to satisfy transit observation at visible wavelengths and obscure sufficiently the H 2 O molecular absorption features between 1.1 µm and 1.7 µm.

However, only the highest metallicity case considered (10000×solar) brings the simulated spectra into closer agreement with transit depths at 3.6 µm and 4.5 µm indicating a high contribution of CO/CO 2 in GJ1214b's atmosphere. We also evaluate the impact of aggregate growth in our simulations, in contrast to spherical growth, and find that the two growth modes provide similar transit signatures (for D f =2), but with different particle size distributions. Finally, we conclude that the simulated haze particles should have major implications for the atmospheric thermal structure and for the properties of condensation clouds.

INTRODUCTION

Super-Earth and mini-Neptune size planets constitute a large fraction of the observed exoplanet population, therefore are the subject of intense investigation. Transit observations of such planets reveal a diversity in the depth of spectral features anticipated at near IR wavelengths from molecular absorption, which is attributed to the presence of suspended particulate matter in their atmospheres (Crossfield & Kreidberg 2017). Such het-ical hazes in the atmospheres of super-Earths/mini-Neptunes using detailed models of atmospheric chemistry and haze microphysics, and evaluate how such components can help interpret the available observations. The most well studied example of such an atmosphere is the case of exoplanet GJ 1214 b, for which multiple ground based and space-borne observations reveal a remarkably flat spectrum extending from Visible to IR (see Angerhausen et al. 2017, for an overview of available transit observations). A high precision evaluation of transit depth at visible wavelengths is prevented by the temporal and spatial inhomogeneities of the GJ 1214 M-dwarf stellar emission (Rackham et al. 2017;Mallonn et al. 2018). Nevertheless, the most accurate constraints at visible from ground based observations with the Large Binocular Telescope (Nascimbeni et al. 2015), in combination with the high-precision Hubble Space Telescope (HST) observations between 1.1 and 1.7 µm (Kreidberg et al. 2014), and the Spitzer measurements at 3.6 and 4.5 µm (Fraine et al. 2013) reveal a quasi-flat spectrum, that restricts the characterisation of the atmospheric composition. Simulated transit spectra assuming only gaseous components are inconsistent with the observations even when high solar metallicities are assumed or even pure H 2 O/CO 2 atmospheres, hence making the presence of the heterogeneous opacity necessary (Kreidberg et al. 2014). Although the almost featureless transit spectrum of GJ 1214 b could be fitted by a planetary body with no substantial atmosphere, such a scenario seems unlikely as the measure radius and mass of the planet provide a bulk density (ρ∼2 g cm -3 ) that is well below the typical rock/metal composition limit. Mass and radius measurements cannot uniquely constrain the composition of the planet, however, thermal evolution models of the planet interior suggest that a few percent of the planet mass should be due to a H/He envelope to explain the measured mass density (Lopez & Fortney 2014;Lozovsky et al. 2018). Thus, the presence of an atmosphere on GJ 1214 b is rather probable.

Preliminary studies for the properties of clouds and photochemical hazes in the atmosphere of GJ 1214 b, suggested that both components can provide a flat spectrum (Morley et al. 2013(Morley et al. , 2015)). Photochemical hazes are expected to form in the upper atmosphere (p<10µbar) and coagulate to larger particles as they sediment to the lower atmosphere, while clouds are expected to form at higher pressures depending on the solar metallicity and temperature conditions. However, the interpretation of the observations with condensates of KCl and ZnS composition (the most likely cloud condensation candidates for the anticipated atmo-spheric conditions) require high metallicity conditions 100 (1000×solar), as well as, significantly reduced sedimen-101 tation velocities for the formed cloud particles. On The broken lines present the condensation curves for ZnS (dashed), KCl (dash-dotted), and Na2S (dash-triple-dotted) assuming thermochemical equilibrium (Morley et al. 2012), while the blue curves present the assumed atmospheric mixing profile for each case using input from the GCM models (Charnay et al. 2015a). The right side panels present the mean molecular weight (black) and scale height (blue) of the atmosphere based on our disequilibrium chemical composition.

For the evaluation of the photochemical haze proper- calculations. Finally, we assume isothermal profiles in With the above inputs we calculate the chemical composition of GJ 1214 b under different metallicity assumptions (Fig. 3). Increasing the atmospheric metallicity leads to an increase in the atmospheric mean molecular weigh with a concurrent decrease in the atmospheric scale height (Fig. 1). The mixing ratios of main atmospheric components in the middle atmosphere (1 mbar) are shown in 9.1(-14) 1.1(-12) 2.6(-12) 7.4(-12) 2.9(-12) 1.5(-13) 4.8(-13) 8.9(-12) 7.4(-11) 3.5(-11) NH3

3.9(-12) 3.9(-12) 2.8(-12) 1.7(-12) 9.3(-13) 1.3(-11) 2.6(-11) 4.0(-11) 3.4(-11) 6.3(-12) HCN 2.9(-12) 1.0(- 12135.8(-16) 4.7(-15) 6.3(-14) 1.3(-12) 2.9(-12) 6.2(-15) 1.0(-13) 2.0(-12) 3.9(-11) 6.9(-11) CO 3.7(-24) 3.7(-19) 1.2(-17) 1.5(-14) 1.0(-12) 6.9(-17) 2.4(-14) 2.4(-13) 2.8(-12) 3.1(-11) H2O

2.6(-11) 2.6(-11) 1.8(-11) 1.4(-11) 3.7(-12) 1.5(-10) 1.5(-10) 1.7(-10) 1.8(-10) 5.2(-11) Soot(1%) 1.9(-13) 9.2(-14) 5.9(-14) 9.3(-14) 1.2(-13) 1.3(-12) 1.2(-12) 7.2(-13) 1.1(-12) 4.7 (-13) Hazes based on sulfur composition are also a possible candidate for the temperature conditions of mini-Neptunes/super-Earths (Zahnle et al. 2016;Gao et al. 2017). However, for the solar elemental compositions we consider C is more abundant than S, thus is likely to provide a higher abundance of soot type hazes. Moreover, a correct representation of sulfur components requires consideration of cloud formation as ZnS and Na 2 S are likely condensates at the conditions under investigation that will limit the abundance of sulfur-based haze precursors reaching the upper atmosphere. Thus, we focus here on the formation of soot-type haze precursors.

The haze formation yield for HD 189733 b was found to range between 0.5% and 10% depending on the atmospheric conditions assumed (Lavvas & Koskinen 2017), while we know that Titan's atmosphere (the most hazy in the solar system) has a haze yield of ∼30% (Lavvas et al. 2011a(Lavvas et al. , 2013)). Thus, for our preliminary estimates of haze mass fluxes we consider a conservative 1% yield in our tabulated values (see Table 2). We use this estimate only as a tool for comparison among the different metallicity cases, as we evaluate further below what would be the required yield to explain the transit observations. We present chemical composition calculations for both limb (calculated under spherical geometry for tangent rays) and disc (dayside stellar flux for incidence angle of µ 0 =0.5) geometry conditions to evaluate what would be the difference in the haze production rate at different locations (given the relatively small, ±100 K, horizontal temperature difference suggested by the GCM we consider the same temperature profile for different geometries). However, we assume that the atmospheric circulation will act towards a homogenisation of the particle distribution, although this is something that needs to be evaluated with a coupled model of atmospheric circulation and haze microphysics. Nev- With the assumed 1% soot formation yield, haze mass 366 fluxes range between ∼2×10 -14 gcm 2 s -1 and ∼10 -13 367 gcm 2 s -1 for limb geometry and between 7×10 -13 368 gcm 2 s -1 and 1.3×10 -12 gcm 2 s -1 for disk geometry, 369 for the different metallicity cases. However, increasing 370 the atmospheric metallicity causes changes in the haze 371 production that are not monotonic (Table 2). Using the 372 100×solar metallicity results as a reference case we find 373 that lower metallicity conditions result in higher haze light this effect (Fig. 6). These times are calculated 505 from:

506 τ S = H < v p > , τ M = H 2 K ZZ , τ C =< n p ∂n p /∂t > (1)
with brackets corresponding to averaging over the par- the rate of increase in the average particle size becomes 524 progressively smaller with increasing metallicity (Fig. 5).

525

Our simulations show that at 1 mbar and for a haze small uncertainty and we wish to utilise the measurements at shorter wavelengths for the characterisation of the haze properties.

The simulated spectra of clear atmospheres demonstrate how the increase in metallicity makes the transit signatures more shallow due to the drop of the atmospheric scale height. However, even for the high- Even if low metallicity cases do not apply to the atmosphere of GJ 1214 b, it is useful to highlight the variation of photochemical haze contribution in the observed spectra for consideration in other planetary cases (Crossfield & Kreidberg 2017). At low metallicities, we saw that the atmospheric scale height is large and mixing time scales are low. Thus, haze particles remain at high altitudes with a large abundance and affect significantly the observed transit spectrum at visible wavelengths, even for very low formation yields (∼0.01% for solar metallicity). As metallicity increases, haze opacity in the upper atmosphere decreases and for a given haze mass flux the pressure probed during transit moves to higher pressures, since photons can penetrate to deeper layers before haze opacity sufficiently increases to block them (Fig. 7). For metallicities at and above 100×solar, a higher haze formation yield needs to be considered in order to bring the simulated spectra close to the observations. A mass flux of the order 10 -11 g cm -2 s -1 provides enough opacity in the visible/near-IR to bring the transit spectrum close to the observations. Such a mass flux suggests a haze formation yield of ∼10% -20%. However, as we discuss below, contributions from the photolysis of CO/CO 2 could further enhance the haze mass fluxes in which case a common ∼10% for all high metallicity cases (above 100× solar) is sufficient to reproduce the observations.

The observed variation of the transit signature among the two Spitzer wavelengths, with the transit depth at 4.5 µm higher than at 3.6 µ by ∼4σ suggests that the higher metallicity end provides the most representative case among those we consider. In our simulations the increase of CO 2 with increasing metallicity reduces the high contrast between the two bands characteristic of low metallicities and brings them closer to the observed behaviour at the 10000×solar metallicity case. However, even in this case the simulated ratio of transit at the two

707

The combined contributions of photochemistry that 708 leads to the inception of particles, heterogeneous chem-709 istry on the particle surface, and particle collisions, de-710 fine the primary particle radius (Lavvas et al. 2011b).

711

Typical values of primary particle radii from atmo-712 spheres of our solar system are of the order of 10s of 713 nm, e.g. 10 nm for the aggregates in Pluto's atmo- 853 Thus, we did not consider it here. 2. Solid lines present the case of nominal haze production by photolysis of CH4, N2 and of their main photochemical products, while dashed lines consider the additional contributions from CO and CO2 photolysis.

a factor of ∼3 between the two extreme cases of solar and 10000×solar metallicity. This is a clear demonstration of the fact that photochemical products are dominantly photon limited, as long as, the abundance of precursors is not critically affected by their photoly- ), with production rates varying according to 899 the energy source and the temperature conditions 900 (Hörst et al. 2018;He et al. 2018) become more carbonaceous at these conditions (Fig. 13).

995

The CO/CO 2 contributions bring the relative haze the end products of the photochemistry, while our theoretical estimates are based on the first steps of the atmospheric photochemistry. These two extremes are related by the haze formation yield. The qualitative consistency between our theoretical simulations and the experimental results verify the appropriateness of this approach.

However, the comparison also demonstrates that utilising the photolysis mass fluxes provides a more robust estimation of the haze production, instead of scalings based on the abundances of individual precursors. This is readily demonstrated by observing that abundances across metallicity cases demonstrate a monotonic variation (e.g. CH 4 and HCN in Fig. 3) that would result in monotonic haze production yields across metallicities, inconsistent with the experimental results.

We highlight that the above discussion applies only to the relative production yields. The experiments reveal that absolute yields increase with decreasing temperature for the 100× and 1000× metallicity cases, while the opposite behaviour was observed for the 10000×solar metallicity case (He et al. 2018). The absolute haze production rates for the latter case were measured to raise from 0.01 mg hr -1 at 400 K to 0.013 mg hr -1 at 600 K. The ratio of 1.3 between these to values is tantalisingly close to the value of 1.35 we estimate for the ratio of photolysis rates, J 600K /J 400K , of a pure CO 2 gas at these two temperature limits (Venot et al. 2018), assuming the spectral density of the UV lamp (Fig. 2). Given that CO 2 is the main gas component at this metallicity, it is logical to correlate the temperature dependence of the CO 2 cross section with the observed variation of the haze production. At lower metallicities, the contribution of CO 2 is smaller therefore its impact on the haze pro-Figure 16. Homogeneous (red) and heterogeneous nucleation rates for KCl. For the heterogeneous rates we consider our simulated haze particle distribution (spheres) for the 10000×solar metallicity case at 800 K and 0.01 bar. Different curves correspond to various values for the contact angle, θ, between the substrate (haze particle) and the condensed phase, expresses as µ=cos(θ).

duction is smaller. However, the reason for the inversion On the contrary, at longer wavelengths where gas absorption decreases, haze opacity dominates. Thus, as with the case of hot-Jupiter hazes, we find that the presence of such particles in the atmosphere could significantly affect the thermal structure. Our calculations show that absorption of stellar radiation by the particles results in the same energy deposition rate as for the main gas absorbers. For the 10000×solar metallicity that is closer to the observed spectra, CH 4 is the dominant gas absorber followed by H 2 O. Our calculations suggest that the haze contribution in the energy deposition is similar to that of CH 4 and at pressures lower than 0.1 mbar dominates over all gas contributions up to 1 µbar (Fig. 15). Previous studies, focusing on the impact of cloud particles on the energy balance found that they could significantly affect the atmospheric thermal structure (Charnay et al. 2015b;Roman & Rauscher 2019).

Soot composition particles are expected to have a larger impact as they demonstrate higher absorptivity than the cloud composition components. However, the extent of this impact will depend on how efficiently haze particles are consumed as condensation nuclei in cloud formation.

Our preliminary estimates for the impact of nucleation demonstrate that haze particles would efficiently act as nucleation sites. Our calculations for the condensation of KCl that is expected to condense at higher altitudes compared to ZnS (see Fig. 1199

102

  the contrary, photochemical hazes could explain the ob-103 served flatness of the HST observations at lower metal-104 licities (50×solar) and without the requirement of re-105 duced sedimentation (Morley et al. 2015). 106 Studies with general circulation models (GCM) sug-107 gest that atmospheric dynamics could uplift particles in 108 the atmosphere of GJ 1214 b (at pressures higher than 109 ∼0.1mbar), with increasing efficiency as the metallic-110 ity increases above solar (Charnay et al. 2015a). Under 111 the assumption that cloud particle sizes are constant 112 with height a particle radius of 0.5 µm provided the best 113 match to the HST observations (Charnay et al. 2015b). 114 Subsequent investigations focusing on the micro-115 physics of cloud formation (Gao & Benneke 2018) 116 demonstrate that KCl and ZnS clouds could match 117 the available observations only at high metallicities 118 (1000×solar) and under the assumption of strong at-119 mospheric mixing (K ZZ =10 10 cm 2 s -1 ). However, such 120 an efficient mixing is not supported by the GCM results 121 (K ZZ =10 7 -10 9 cm 2 s -1 from Charnay et al. 2015a), 122 while heterogeneous nucleation on the surface of pho-123 tochemical hazes, not yet evaluated, could affect the 124 formation of the condensates and modify the result-125 ing cloud particle distribution (Gao & Benneke 2018).

126A

  similar qualitative conclusion for the shortcomings 127 of condensates to explain the GJ 1214 b transit ob-128 servations was derived from Ohno & Okuzumi (2018). 129 These results suggest that photochemical hazes could 130 potentially provide a more realistic interpretation. 131 Previous work on the microphysics of photochemi-132 cal haze formation on sub-Neptune size planets demon-133 strated how the variable degree of haze production can 134 result to different levels of transit spectra flattening 135 (Kawashima & Ikoma 2018) and how aggregate growth 136 can modify the haze properties (Adams et al. 2019). 137 Here we explore further the details of the microphysi-138 cal description and evaluate the haze properties at dif-139 ferent conditions of solar metallicity. We furthermore 140 evaluate the differences between spherical and aggre-141 gate growth and discuss the possible implications of 142 the formed hazes on the atmospheric structure. Our 143 work addresses the general properties of photochemical 144 hazes on super-Earth/mini-Neptunes atmospheres and 145 for that reason we compare our results with labora-146 tory experiments simulating the photochemical haze for-147 mation at such conditions (He et al. 2018; Hörst et al.148 2018).

Figure 1 .

 1 Figure 1. Atmospheric structure assumed for GJ 1214 b at different metallicities. Left side panels present the thermal structure (black solid lines) based on Gao & Benneke (2018).The broken lines present the condensation curves for ZnS (dashed), KCl (dash-dotted), and Na2S (dash-triple-dotted) assuming thermochemical equilibrium (Morley et al. 2012), while the blue curves present the assumed atmospheric mixing profile for each case using input from the GCM models(Charnay et al. 2015a). The right side panels present the mean molecular weight (black) and scale height (blue) of the atmosphere based on our disequilibrium chemical composition.

Figure 2 .

 2 Figure 2. Stellar emission from GJ 1214 assumed in the calculations. The red line shows the high-resolution spectrum based on the MUSCLES database, and the black line depicts the spectrum mapped to the resolution of our model (see text). The inset presents the UV light lamp used for laboratory haze formation for comparison (He et al. 2018).

Figure 3 .

 3 Figure 3. Chemical composition of GJ 1214 b for the five metallicity cases and for disk geometry. The top panels present the variation of the main composition (broken line corresponds to atomic hydrogen) and the bottom panels the corresponding photochemical products of hydrocarbons and oxygen species.

Figure 4 .

 4 Figure 4. Photolysis mass fluxes from various molecules for different metallicity cases.

  365

374

  mass fluxes by factors of 1.8 and 1.7 at 1× and 10× solar 375 metallicity, respectively. At 1000×solar metallicity the 376 haze production increases again by a factor of 1.5 before 377 dropping by a factor of 0.65 at 10000×. The reasons 378 for these variations are due to the changes in the main atmospheric composition at each metallicity case and 380 their implications on the photolysis rates. 381 At low metallicities the catalytic destruction of H 2 382 through interaction with OH, produced in the photodis-383 sociation of H 2 O, leads to the formation of a large 384 abundance of atomic hydrogen in the upper atmosphere 385 (Moses et al. 2011). The latter is a major pathway for 386 the breakup of CH 4 (H + CH 4 → H 2 + CH 3 ) and the 387 formation of higher order hydrocarbons. Therefore, for 388 the solar metallicity case, at pressures lower than 1 µbar 389 methane is lost and C 2 -type hydrocarbons are the main 390 hydrocarbon precursors for photochemical haze forma-391 tion (Fig. 3). As metallicity increases to 10× and 100× 392 solar, the atmospheric pressure where the catalytic de-393 struction of H 2 takes place moves to lower pressures 394 because the abundance of H 2 O increases and UV pho-395 tons are consumed more efficiently in the upper atmo-396 sphere leading to smaller penetration in the atmosphere. 397 Therefore, more methane survives in the upper atmo-398 sphere and the production of higher order hydrocarbons 399 is reduced. Hence, the photolysis contributions of higher 400 order hydrocarbons decrease and that of CH 4 increases 401 (Table 2 & Fig. 4). Moreover, as metallicity increases, 402 UV photons leading to H 2 O photolysis are absorbed by 403 other species as well, leading effectively to a reduction 404 of the atomic hydrogen abundance. This reduction of 405 atomic hydrogen in the upper atmosphere is more promi-406 nent for the 1000× and 10000× solar metallicity cases 407 (Fig. 3). At these conditions, the relative contribution 408 of direct photolysis of CH 4 becomes more important and 409 provides the local increase in the haze production rate in 410 the 1000×solar metallicity case. At 10000×solar metal-411 licity, the CH 4 abundance is reduced, while the opacity 412 of other competing species such as CO and CO 2 is higher 413 leading to a reduction in methane photolysis (as well as 414 that of other haze precursor species) and an overall de-415 crease of haze production (Fig. 4). Below, we further 416 discuss these results in the context of laboratory exper-417 iments.

Figure 5 .

 5 Figure 5. Average radius and corresponding number density of haze particles in the atmosphere of GJ 1214 b, under different assumptions of haze mass flux and metallicity.

Figure 6 .

 6 Figure6. Characteristic times for settling, mixing, and coagulation (collisions) for the haze particles of GJ 1214 b's atmosphere, for different metallicity cases. The haze mass flux is 10 -13 gcm -2 s -1 .

507

  ticle size distribution. At solar metallicity, particle col-508 lisions have the smallest characteristic time leading to 509 a rapid growth of the average particle size. Only be-510 low ∼1bar, atmospheric mixing due to the strong inver-511 sion assumed in the eddy profile, dominates over colli-512 sions and the particles are rapidly transferred towards 513 the lower boundary, thus explaining the rapid drop on 514 the particle number density observed for all metallicity 515 cases at that location. At higher metallicities, as the 516 mean molecular weight increases and the atmospheric 517 scale-height decreases, the characteristic time for atmo-518 spheric mixing in the upper atmosphere decreases. Thus 519 even if the eddy mixing profile is the same for the 10×, 520 100×, and 1000× solar metallicity cases, the character-521 istic time of atmospheric mixing becomes progressively 522 smaller and particles are subject to less collisions. Thus, 523

526Figure 7 .

 7 Figure 7. Observed (black crosses) and simulated (color lines) transit depth of GJ 1214 b. Each panel presents a different metallicity case and each color line a variation on the assumed haze mass flux. The points and associated numbers on the right of each panel present the pressure (in µbar) probed at each wavelength. Note that for clarity the pressure range plotted in each panel is different.

  est metallicity case considered (10000xsolar) a clear atmosphere (considering only the gas phase abundances) cannot reproduce the observed flatness as the transit depth at visible is always short of the observations(Nascimbeni et al. 2015), while the H 2 O bands provide much deeper troughs than the observations reveal in the near IR(Kreidberg et al. 2014). Thus, a heterogeneous opacity is necessary to explain the observations.

Figure 8 .Figure 9 .

 89 Figure 8. Comparison of spherical (blue) and aggregate (red & green) growth of haze particles for 1× and 10000× metallicity cases. Each broken line corresponds to a different primary particle radius. For aggregates the red lines correspond to the average radius of the equivalent mass spherical particle and the green lines to the fractal structure average radius. The corresponding average particle density is shown on the right panels (it does not depend on the radius-type averaging). Note that the assumed mass fluxes for each metallicity case are different and correspond to those providing results close to the observed transit spectra.

  714 sphere (Gladstone et al. 2016) and 40 nm for Titan's

Figure 10 .Figure 11 .

 1011 Figure10. Particle size distribution for spherical (black) and aggregate growth (red, 10 nm primary particles) for the 10000×solar metallicity case and for a haze mass flux of 10 -11 gcm -2 s -1 . Each line style corresponds to a different pressure level as shown.

Figure 12 .(

 12 Figure12. Comparison of transit spectra for spherical and aggregate shape haze simulations (see Fig.8) for the 1× and 10000×solar metallicity cases. Note that spectra are smoothed for clarity.

Figure 13 .

 13 Figure 13. Fractional abundance of different elements in the photolysis mass fluxes presented in Table2. Solid lines present the case of nominal haze production by photolysis of CH4, N2 and of their main photochemical products, while dashed lines consider the additional contributions from CO and CO2 photolysis.

Figure 14 .

 14 Figure14. Relative haze production from simulations (lines) and laboratory experiments (asterisks) at different metallicities (see text). The black line corresponds to the nominal haze precursors (CH4 & N2 photochemical products), while the dotted and dash-dotted lines present additional contributions from the photolysis of CO and CO2, respectively. All curves are normalised to the 100×solar metallicity case. Pie charts at the bottom present the main gas composition for each case.

996

  production close to the metallicity behaviour seen at 997 600 K, but they also increase further the production 998 at the 1000×solar metallicity case. A naive interpre-999 tation of the experimental results at the two temper-1000 atures would suggest that the relative contribution of 1001 CO/CO 2 -produced hazes is higher at 600 K than at 400 1002 K. Although such a behaviour may partially contribute 1003 to the observed behaviour, these relative yields have to 1004 be examined through the prism of the main gas com-1005 position. The main difference of the gas composition at 1006 the two temperatures studied in the laboratory experi-1007 ments at 10000×solar metallicity, is that at 400 K there 1008 is ∼3× more H 2 O than at the equilibrium composition 1009 at 600 K, that could impose a significant reduction on 1010 the haze formation, again through photolysis screening 1011 of other species, as well as, by oxidizing reactions. In 1012 our corresponding disequilibrium simulations the H 2 O 1013 abundance (1.2%) is closer to the 600 K case, for which 1014 we get a similar relative production. 1015 In addition, differences in the chemical rates imposed 1016 by the different temperature conditions could further 1017 affect the haze production. We note that experimental 1018 and theoretical results correspond to the different steps 1019 of the photochemical haze production: the experiments 1020 measure the abundance of the formed haze particles i.e.

Figure 15 .

 15 Figure 15. Energy deposition in the atmosphere of GJ 1214 b from the absorption of stellar radiation. Here we compare the haze contribution with the main gaseous absorbers.

1055

  of the haze yield with temperature is not clear and has to 1056 be sought in the chemical pathways leading to the haze 1057 formation. Such an investigation necessitates a detailed 1058 comparison with the experiments at identical conditions 1059 (gas mixtures/ energy input) to identify the role of each 1060 process on haze production, and which will further ben-1061 efit from comparison with the experimentally derived 1062 gas phase products (He et al. 2019). Such a study goes 1063 beyond our goals for the current investigation. How-1064 ever, the conclusion we derive from this comparison of 1065 relative production yields is that the theoretical simu-1066 lations capture the qualitative behaviour identified in 1067 the experiments and vice versa the experiments provide 1068 a picture of haze formation representative of theoreti-1069 cal anticipations, indicating promising results from this 1070 venue of investigation. 1071 We now turn our attention to the implications of the 1072 haze particles on the atmosphere. We find that our 1073 simulated haze distributions (of either spherical or ag-1074 gregate shape) do not impose major modifications to 1075 the radiation field at UV wavelength, therefore do not 1076 have a major impact on the simulated chemical abun-1077 dances. This occurs because for the sub-Neptune condi-1078 tions investigated here gases provide a larger UV opac-1079 ity than haze, under all metallicity conditions. For 1080 example, at solar metallicity conditions for which we 1081 can compare with previous evaluations for hot-Jupiters 1082 (Lavvas & Koskinen 2017), H 2 O, NH 3 , and CH 4 have 1083 higher abundances for sub-Neptune conditions, therefore dominate the UV opacity.

  1 and Gao & Benneke 2018), demonstrate haze particles would readily lead to the formation of cloud particles at very low super-saturations (Fig.16). Even for small values of the contact angle between the haze particle and the condensed phase formed on its surface, a parameter that is currently unknown, the heterogenous nucleation rate dominates over the homogeneous nucleation rate at low super-saturations. Currently in the simulations of cloud formation in mini-Neptunes/super-Earths (Ohno & Okuzumi 2018; Gao & Benneke 2018) KCl condensation is assumed to start through homogeneous nucleation, while ZnS is formed heterogeneously on the surface of the KCl cloud particles. Thus, the different population of nucleation sites imposed by the presence of the photochemical hazes will have implications for the properties of all subsequent cloud particle distributions. Therefore, our results demonstrate that a coupled picture between photochemical hazes and cloud formation is necessary.Our simulations provide a disk-average picture of haze properties for the atmosphere of GJ 1214 b, which will be further modulated by the atmospheric circulation. Although the assumed K ZZ profile in our study does take into consideration the impact of advection on the vertical direction, horizontal re-that these modifications depend on the complex inter-1187 play between the dominant gaseous abundances for each 1188 metallicity case and their interaction with the incoming 1189 stellar radiation. Comparison of our simulations with 1190 laboratory experiments of photochemical haze produc-1191 tion reveals a qualitatively consistent picture. On a sec-1192 ond degree further variations will be imposed by changes 1193 on the chemical pathways leading to haze formation that 1194 are currently unknown, and which will define the abso-1195 lute haze formation yield. These are aspects that need to 1196 be investigated in the future through combined theoreti-1197 cal and experimental approaches under well constrained 1198 conditions.

Table 1 .

 1 Main atmospheric composition at 1 mbar for different metallicity cases according to the disequilibrium chemistry calculations. Read a(b) as a×10 b .

		Species	1x	10x	100x	1000x	10000x
		H2	0.835	0.822	0.706	0.341	7.08(-2)
		He	0.163	0.164	0.167	0.111	2.14(-2)
		H2O	7.13(-4) 7.12(-3) 5.58(-2) 7.77(-2) 1.22(-2)
		CH4	5.94(-4) 6.03(-3) 4.64(-2) 6.74(-2) 1.05(-2)
		N2	3.37(-5) 7.79(-4) 9.52(-3) 6.39(-2)	0.123
		NH3	1.15(-4) 3.26(-4) 3.66(-4) 4.10(-4) 5.39(-5)
		CO	5.16(-7) 1.56(-5) 1.35(-2)	0.274	0.651
		CO2	2.11(-10) 2.16(-7) 1.61(-3) 6.43(-2)	0.111
	252	of 1.3×10 -14 erg cm -2 s -1 Å-1 reaching the Earth
	253	(Youngblood et al. 2016) . This value can be used to
	254	estimate the EUV emission at broad bins between 100
	255	Å and 1170 Å (Linsky et al. 2014). The spectral den-
	256	sity within each EUV bin is unknown, thus we assume
	257	a solar-type spectral distribution. A similar combina-
	258	tion of X-ray observations and simulations provides the
	259	spectrum at shorter wavelenghts (Fontenla et al. 2016).
	260	GJ 1214 is at 14.6 pc, which is sufficiently distant that
	261	there exist large uncertainties in the observed spectrum.
	262	However, observations of a similar spectral type star, GJ
	263	876 (M5, T ef f = 3062 K, log g(cms -2 ) = 4.93) at 4.7 pc
	264	provide an improved accuracy spectrum. Thus, we used
	265	the spectral density of GJ 876 to estimate the spectral
	266	emission of GJ 1214 for regions where noise dominated
	267	the observations, particularly at the Ly-α wings and
	268	up to ∼3000 Å. At longer wavelengths the MUSCLES
	269	spectrum is based on observations with HST/STIS and
	270	HST/COS combined with PHOENIX stellar emission
	271	models.
	272	At the lower boundary (10 3 bar) we consider abun-
	273	dances derived from the CEA thermochemical equilib-
	274	rium model (McBride et al. 2002). The various metallic-
	275	ity cases we consider result in different main atmospheric
	276	compositions that reflect on the mean molecular weight
	277	and the corresponding atmospheric viscosity. These pa-
	278	rameters are important for the simulation of molecu-
	279	lar diffusion and settling velocities of haze particles and
	280	are calculated self-consistently as a function of pressure
	281	within our model. The mean molecular weight is calcu-
	282	lated from our chemical composition calculations, while
	283	for the atmospheric viscosity we use the corresponding
	284	state method for gas mixtures, taking into account the
	285	impact of pressure on the gas viscosity at p>10 bar
		(Poling et al. 2001).

286

3. HAZE PRODUCTION

Table 1

 1

	for each

326

Table 2 .

 2 Photolysis mass fluxes (g cm -2 s -1 ) in the upper atmosphere of GJ 1214 b under different assumptions of solar metallicity. Soot mass fluxes are based on the contributions of CH4, NH3, HCN, C2H2, C2H4, and N2 scaled by a yield of 1%.

			Metallicity -Limb			Metallicity -Disk
	Species	1×	10×	100×	1000×	10000×	1×	10×	100×	1000×	10000×
	CH4

  wavelength bands is not as close as in the observations

	635
	636	suggesting a further enhancement of CO 2 relative to our
	637	calculations.
	638	6. AGGREGATION
	639	So far we assumed that the haze particles grow as
	640	spheres. However, aggregation is a physical consequence
	641	of solid particle collisions, and is likely to occur for the
	642	photochemical hazes we consider here. The onset of
	643	aggregate formation occurs when mass addition from
	644	the gas phase is negligible compared to the mass added
	645	through particle collisions. Vice versa, aggregated par-
	646	ticles can grow to a spherical shape if the rate of mass
	647	addition by gas deposition (heterogeneous processes) is
	648	larger than the rate of mass addition due to coagula-
		tion, as demonstrated by experiments and simulations
	670
	671	resulting in higher coagulation rates, hence, faster par-
	672	ticle growth. Moreover, aggregates with structures typ-
	673	ical of those found in Titan's atmosphere (D f =2, see
	674	below) have sedimentation velocities representative of
	675	their primary particle size in the free molecular regime
	676	(Cabane et al. 1993; Rannou et al. 2010; Lavvas et al.
	677	2010). Thus, the rapid growth of aggregates will not
	678	be limited by an increase of their settling in the upper
	679	atmosphere, as would be the case for spherical parti-
	680	cles. Therefore, we explore here the haze properties for
		aggregates.

649

(see

Lavvas et al. 2011b, and references therein)

. Such 650 a shape transition is more feasible when particles have 651 a small size, which is why in our previous study of hot-652 Jupiters we considered only spherical growth. Our simu-653 lations suggested that the particle size required to match 654 the transit observations of HD 189733 b was of the or-655 der of few nm, at which size heterogeneous reactions on 656 the particle surface could readily preserve the sphericity 657 of the particles (Lavvas & Koskinen 2017; Lavvas et al. 658 2011b). On the contrary, our simulations for GJ 1214 b, 659 depending on the assumed metallicity, show that larger 660 particle sizes are possible to affect the transit signature. 661 Larger particles in the upper atmosphere are mainly 662 characteristic of low metallicity conditions, which ap-663 pears inconsistent with the near IR transit signatures. 664 However, aggregate particles have different optical and 665 microphysical properties (in terms of their collisional 666 efficiency and aerodynamic drag) from spherical parti-667 cles, which may result to a different transit depth sig-668 nature. For example, an aggregate particle has a larger 669 cross section than the same mass spherical equivalent, 681 An overview of the properties of aggregate particles 682 can be found in previous studies on Titan's atmosphere, 683 where hazes are known to be of aggregate structure 684 (see review by West et al. 2014, and references there 685

  . For experiments

			970	For example, at 1000×solar metallicity H 2 O and CO 2
	919	is defined by disequilibrium chemistry (Table 1). On	971	have significant abundances in the experimental mix-
	920	the contrary, the experiments performed at the brack-	972	tures at 400 and 600 K respectively, which are replaced
	921	eting 400 K and 600 K temperature cases had a main	973	by H 2 and CO in our calculations. The latter two gases
	922	gas composition based on thermochemical equilibrium	974	do not intervene in the photolysis of hydrocarbons con-
	923	conditions at 1 mbar pressure (He et al. 2018). Never-	975	trary to the former two gases that have major absorption
	924	theless, our calculated composition at 100× and 1000×	976	bands at UV wavelengths and will screen the photolysis
	925	solar metallicity cases are not very different from those	977	of methane and other hydrocarbons resulting in smaller
	926	of the experiments (see pie charts in Fig. 14). At 100×	978	haze production.
	927	the disequilibrium CH 4 mixing ratio at 500 K (4.6%) is	979	At 10000×solar metallicity, our calculations show a
	928	very close to the experimental value used (4.5% at 400	980	clear decrease in the haze production that is consistent
	929	K and 3.4% at 600 K), and the rest of the main compo-	981	with the experimental results at 400 K. However, our es-
	930	sition is similar between our calculations and the exper-	982	timated haze production is based only on hydrocarbons
	931	iments with only difference a small contribution (∼1%)	983	and N-containing species (N 2 , NH 3 , HCN), while the
	932	of N 2 in our calculations. At 1000× our CH 4 mixing ra-	984	experimental conditions do not include any CH 4 . This
	933	tio (6.7%) is in between the two equilibrium abundances	985	characteristic suggests that formation of photochemical
	934	(11% at 400 K and 1.7% at 600 K). However the main	986	hazes from the photolysis of CO/CO 2 is also possible
	935	gas composition in our simulation is based on H 2 (34%)	987	(He et al. 2018; Hörst et al. 2018). If we consider the
	936	and CO (27%), while for the experiments the main equi-	988	possible contribution of carbon from the photolysis of
	937	librium composition changes from H 2 O (56%) at 400 K	989	CO in the theoretical haze mass flux estimates (dotted
	938	to H 2 (42%) and CO 2 (20%) at 600 K. These changes		curve in Fig. 14) we derive a higher haze production,
	939	in the main composition of our simulations occur due to
	940	quenching in the lower atmosphere by the atmospheric
	941	mixing (Fig. 3). However the relative contributions of C-
	942	H/C-O species are qualitatively similar between our cal-
	943	culations (6.7/33.8) and the experiments (11/10 at 400K
	944	and 1.7/21.9 at 600 K). This picture drastically changes
	945	at 10000×solar metallicity case for which the disequi-
	946	librium composition includes ∼1% CH 4 , while for the
	947	equilibrium conditions methane is a minor species not
	948	included in the experimental gas mixture. In addition,
	949	the main composition in the disequilibrium conditions is
	950	based on CO (65%) with contributions from CO 2 (11%)
	951	and N 2 (12%), while at equilibrium CO 2 (67%) is the
	952	main gas component (Fig. 14). There are also differ-
	953	ences in the spectral density of the UV light used in the	901
	954	experiment and that of GJ 1214, that impose further	902	performed under UV irradiation, which are more rel-
	955	reasons for differentiation in the results. Ly-α (121.6	903	evant to the photochemically produced hazes stud-
	956	nm) dominates the UV stellar spectrum, while the labo-	904	ied here, particle production for each metallicity case
	957	ratory UV lamp has a stronger output near 160 and 230	905	dropped when temperature increased from 400 to 600 K
	958	nm relative to 121.6 nm. Notwithstanding these differ-	906	(He et al. 2018). For each temperature, particle produc-
	959	ences between experimental and theoretical derivations	907	tion among different metallicity cases showed a different
	960	we can identify some qualitative similarities.	908	dependence (Fig. 14): at 400 K the relative haze pro-
	961	For both experiments and theoretical calculations in-	909	duction was slightly higher at 1000× compared to 100×
	962	creasing the metallicity from 100× to 1000×solar results	910	solar metallicity, and dropped by a factor of 2 at 10000×,
	963	in higher haze production (Fig. 14). Although the pho-	911	while at 600 K a monotonic increase was observed with
	964	tolysis mass fluxes from the disequilibrium chemistry	912	increasing metallicity.
		suggest a stronger increase than the experimental re-	913	A direct comparison with these experimental results
			914	is not straight forward as we can compare only relative
			915	yields among the different metallicity cases considered.
			916	Moreover, our simulations are representative of the pho-
	969		917	tochemistry occurring at 500 K in the upper atmosphere

965

sults at 400 and 600 K, qualitatively both theory and 966 experiments provide the same picture, and the observed 967 differences are likely related to the differences in the gas 968 abundances and UV spectral density identified above. 990 which becomes even higher if we also consider the pho-991 tolysis of CO 2 (dash-dotted curve). In addition, the 992 fractional C-abundance increases with these contribu-993 tions at high metallicity cases, suggesting that the hazes 994

Note that in fig. 2a of Rannou et al. (2016) the assignments of D f are mistakenly inverted.

We thank Prof. P. Rannou for his comments on 1200 the impact of D f on the aggregate optical properties.

1201 This work was supported by the Programme National 1202 de Planétologie (PNP) of CNRS/INSU, co-funded by 1203 CNES. M.S. was supported by NASA Headquarters un-1204 der the NASA Earth and Space Science Fellowship Pro-1205 gram -Grant 80NSSC18K1248. AGM acknowledges the 1206 support of the DFG priority program SPP 1992 "Explor-1207 ing the Diversity of Extrasolar Planets (GA 2557/1-1). 1208 REFERENCES Adams, D., Gao, P., de Pater, I., & Morley, C. V. 2019, 1209 The Astrophysical Journal, 874:61, 14pp 1210 Angerhausen, D., Dreyer, C., Placek, B., et al. 2017, 1211 Astronomy and Astrophysics, 608, A120 1212 Barstow, J. K., Aigrain, S., Irwin, P. G. J., & Sing, D. K. 1213 2017, The Astrophysical Journal, 834, 50 1214 Cabane, M., Rannou, P., Chassefiere, E., & Israel, G. 1993, 1215 Planetary and Space Science (ISSN 0032-0633), 41, 257 1216