Dictyostelium as a Model to Assess Site-Specific ADP-Ribosylation Events.

The amoeba Dictyostelium discoideum is a single-cell organism that can undergo a simple developmental program, making it an excellent model to study the molecular mechanisms of cell motility, signal transduction, and cell-type differentiation. A variety of human genes that are absent or show limited conservation in other invertebrate models have been identified in this organism. This includes ADP-ribosyltransferases, also known as poly-ADP-ribose polymerases (PARPs), a family of proteins that catalyze the addition of single or poly-ADP-ribose moieties onto target proteins. The genetic tractability of Dictyostelium and its relatively simple genome structure makes it possible to disrupt PARP gene combinations, in addition to specific ADP-ribosylation sites at endogenous loci. Together, this makes Dictyostelium an attractive model to assess how ADP-ribosylation regulates a variety of cellular processes including DNA repair, transcription, and cell-type specification. Here we describe a range of techniques to study ADP-ribosylation in Dictyostelium, including analysis of ADP-ribosylation events in vitro and in vivo, in addition to approaches to assess the functional roles of this modification in vivo.

Site-specific ADP-ribosylation of histone H2B in response to DNA double strand breaks.

ADP-ribosyltransferases (ARTs) modify proteins with single units or polymers of ADP-ribose to regulate DNA repair. However, the substrates for these enzymes are ill-defined. For example, although histones are modified by ARTs, the sites on these proteins ADP-ribosylated following DNA damage and the ARTs that catalyse these events are unknown. This, in part, is due to the lack of a eukaryotic model that contains ARTs, in addition to histone genes that can be manipulated to assess ADP-ribosylation events in vivo. Here we exploit the model Dictyostelium to identify site-specific histone ADP-ribosylation events in vivo and define the ARTs that mediate these modifications. Dictyostelium histones are modified in response to DNA double strand breaks (DSBs) in vivo by the ARTs Adprt1a and Adprt2. Adprt1a is a mono-ART that modifies H2BE18 in vitro, although disruption of this site allows ADP-ribosylation at H2BE19. Although redundancy between H2BE18 and H2BE19 ADP-ribosylation is also apparent following DSBs in vivo, by generating a strain with mutations at E18/E19 in the h2b locus we demonstrate these are the principal sites modified by Adprt1a/Adprt2. This identifies DNA damage induced histone mono-ADP-ribosylation sites by specific ARTs in vivo, providing a unique platform to assess how histone ADP-ribosylation regulates DNA repair.

Abi is required for modulation and stability but not localization or activation of the SCAR/WAVE complex.

The SCAR/WAVE complex drives actin-based protrusion, cell migration, and cell separation during cytokinesis. However, the contribution of the individual complex members to the activity of the whole remains a mystery. This is primarily because complex members depend on one another for stability, which limits the scope for experimental manipulation. Several studies suggest that Abi, a relatively small complex member, connects signaling to SCAR/WAVE complex localization and activation through its polyproline C-terminal tail. We generated a deletion series of the Dictyostelium discoideum Abi to investigate its exact role in regulation of the SCAR complex and identified a minimal fragment that would stabilize the complex. Surprisingly, loss of either the N terminus of Abi or the C-terminal polyproline tail conferred no detectable defect in complex recruitment to the leading edge or the formation of pseudopods. A fragment containing approximately 20% Abi--and none of the sites that couple to known signaling pathways--allowed the SCAR complex to function with normal localization and kinetics. However, expression of N-terminal Abi deletions exacerbated the cytokinesis defect of the Dictyostelium abi mutant, which was earlier shown to be caused by the inappropriate activation of SCAR. This demonstrates, unexpectedly, that Abi does not mediate the SCAR complex's ability to make pseudopods, beyond its role in complex stability. Instead, we propose that Abi has a modulatory role when the SCAR complex is activated through other mechanisms.

Cyclical action of the WASH complex: FAM21 and capping protein drive WASH recycling, not initial recruitment.

WASH causes actin to polymerize on vesicles involved in retrograde traffic and exocytosis. It is found within a regulatory complex, but the physiological roles of the other four members are unknown. Here we present genetic analysis of the subunits' individual functions in Dictyostelium. Mutants in each subunit are completely blocked in exocytosis. All subunits except FAM21 are required to drive actin assembly on lysosomes. Without actin, lysosomes never recycle vacuolar-type H(+)-adenosine triphosphatase (V-ATPase) or neutralize to form postlysosomes. However, in FAM21 knockout lysosomes, WASH generates excessive, dynamic streams of actin. These successfully remove V-ATPase, neutralize, and form huge postlysosomes. The distinction between WASH and FAM21 phenotypes is conserved in human cells. Thus, FAM21 and WASH act at different steps of a cyclical pathway in which FAM21 mediates recycling of the complex back to acidic lysosomes. Recycling is driven by FAM21's interaction with capping protein, which couples the WASH complex to dynamic actin on vesicles.

Pseudopod growth and evolution during cell movement is controlled through SCAR/WAVE dephosphorylation.

BACKGROUND: SCAR/WAVE is a principal regulator of pseudopod growth in crawling cells. It exists in a stable pentameric complex, which is regulated at multiple levels that are only beginning to be understood. SCAR/WAVE is phosphorylated at multiple sites, but how this affects its biological activity is unclear. Here we show that dephosphorylation of Dictyostelium SCAR controls normal pseudopod dynamics. RESULTS: We demonstrate that the C-terminal acidic domain of most Dictyostelium SCAR is basally phosphorylated at four serine residues. A small amount of singly phosphorylated SCAR is also found. SCAR phosphorylation site mutants cannot replace SCAR's role in the pseudopod cycle, though they rescue cell size and growth. Unphosphorylatable SCAR is hyperactive-excessive recruitment to the front results in large pseudopods that fail to bifurcate because they continually grow forward. Conversely, phosphomimetic SCAR is weakly active, causing frequent small, disorganized pseudopods. Even in its regulatory complex, SCAR is normally held inactive by an interaction between the phosphorylated acidic and basic domains. Loss of basic residues complementary to the acidic phosphosites yields a hyperactive protein similar to unphosphorylatable SCAR. CONCLUSIONS: Regulated dephosphorylation of a fraction of the cellular SCAR pool is a key step in SCAR activation during pseudopod growth. Phosphorylation increases autoinhibition of the intact complex. Dephosphorylation weakens this interaction and facilitates SCAR activation but also destabilizes the protein. We show that SCAR is specifically dephosphorylated in pseudopods, increasing activation by Rac and lipids and supporting positive feedback of pseudopod growth.

Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis.

WASP and SCAR homologue (WASH) is a recently identified and evolutionarily conserved regulator of actin polymerization. In this paper, we show that WASH coats mature Dictyostelium discoideum lysosomes and is essential for exocytosis of indigestible material. A related process, the expulsion of the lethal endosomal pathogen Cryptococcus neoformans from mammalian macrophages, also uses WASH-coated vesicles, and cells expressing dominant negative WASH mutants inefficiently expel C. neoformans. D. discoideum WASH causes filamentous actin (F-actin) patches to form on lysosomes, leading to the removal of vacuolar adenosine triphosphatase (V-ATPase) and the neutralization of lysosomes to form postlysosomes. Without WASH, no patches or coats are formed, neutral postlysosomes are not seen, and indigestible material such as dextran is not exocytosed. Similar results occur when actin polymerization is blocked with latrunculin. V-ATPases are known to bind avidly to F-actin. Our data imply a new mechanism, actin-mediated sorting, in which WASH and the Arp2/3 complex polymerize actin on vesicles to drive the separation and recycling of proteins such as the V-ATPase.

Activation of the c-Jun N-terminal kinase pathway by MST1 is essential and sufficient for the induction of chromatin condensation during apoptosis.

Chromatin condensation is the most recognizable nuclear hallmark of apoptosis. Cleavage and activation of MST1 by caspases induce chromatin condensation. It was previously reported that, during apoptosis, activated MST1 induced c-Jun N-terminal kinase (JNK) activation and also phosphorylated histone H2B. However, which of these mechanisms underlies MST1's induction of chromatin condensation has yet to be clarified. Here, we report that MST1-mediated activation of JNK is both essential and sufficient for chromatin condensation. MST1 activation did not result in chromatin condensation in mitogen-activate protein kinase kinase 4 (MKK4)/MKK7 double knockout (MKK4/7 DKO) embryonic stem (ES) cells, which genetically lack the ability to activate JNK. On the other hand, constitutively active JNK was able to induce chromatin condensation in MKK4/7 DKO ES cells. In contrast, histone H2B phosphorylation did not correlate with chromatin condensation in wild-type ES cells. Finally, inhibition of JNK as well as inhibitor of caspase-activated DNase blocked chromatin condensation during Fas-mediated apoptosis of Jurkat cells. Taken together, our results indicate that caspase-mediated cleavage of MST1, followed by MST1-mediated activation of the JNK pathway, is the mechanism responsible for inducing chromatin condensation during apoptosis.

Release of RASSF1C from the nucleus by Daxx degradation links DNA damage and SAPK/JNK activation.

Stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) responds to a variety of stress stimuli and controls cell fates such as cell cycle entrance, apoptosis and senescence. Stimuli such as ultraviolet irradiation and chemical reagents that damage genomic DNA induce the activation of the SAPK/JNK signaling pathway. However, it is unclear how the signal arising in the nucleus owing to DNA damage is transmitted to SAPK/JNK in the cytoplasm. Here, we report that the nuclear components Daxx and Ras-association domain family 1C (RASSF1C) link DNA damage to SAPK/JNK activation in HeLa cells. In response to DNA damage, Daxx localized in promyelocytic leukaemia-nuclear bodies (PML-NBs) undergoes ubiquitination and degradation. RASSF1C, a tumor suppressor and newly identified binding partner of Daxx, is constitutively anchored by Daxx in PML-NBs but is released from the nucleus when Daxx is degraded. This released RASSF1C translocates to cytoplasmic microtubules and participates in the activation of SAPK/JNK. Our data define a novel mechanism by which the Daxx-RASSF1C complex in PML-NBs couples nuclear DNA damage to the cytoplasmic SAPK/JNK signaling pathway.