Daniel Monteyne

PI3K Class II α Controls Spatially Restricted Endosomal PtdIns3P and Rab11 Activation to Promote Primary Cilium Function

Summary Multiple phosphatidylinositol (PtdIns) 3-kinases (PI3Ks) can produce PtdIns3P to control endocytic trafficking, but whether enzyme specialization occurs in defined subcellular locations is unclear. Here, we report that PI3K-C2α is enriched in the pericentriolar recycling endocytic compartment (PRE) at the base of the primary cilium, where it regulates production of a specific pool of PtdIns3P. Loss of PI3K-C2α-derived PtdIns3P leads to mislocalization of PRE markers such as TfR and Rab11, reduces Rab11 activation, and blocks accumulation of Rab8 at the primary cilium. These changes in turn cause defects in primary cilium elongation, Smo ciliary translocation, and Sonic Hedgehog (Shh) signaling and ultimately impair embryonic development. Selective reconstitution of PtdIns3P levels in cells lacking PI3K-C2α rescues Rab11 activation, primary cilium length, and Shh pathway induction. Thus, PI3K-C2α regulates the formation of a PtdIns3P pool at the PRE required for Rab11 and Shh pathway activation.

Genetic analysis of a successful repatriation programme: giant Galápagos tortoises

As natural populations of endangered species dwindle to precarious levels, remaining members are sometimes brought into captivity, allowed to breed and their offspring returned to the natural habitat. One goal of such repatriation programmes is to retain as much of the genetic variation of the species as possible. A taxon of giant Galápagos tortoises on the island of Española has been the subject of a captive breeding–repatriation programme for 33 years. Core breeders, consisting of 12 females and three males, have produced more than 1200 offspring that have been released on Española where in situ reproduction has recently been observed. Using microsatellite DNA markers, we have determined the maternity and paternity of 132 repatriated offspring. Contributions of the breeders are highly skewed. This has led to a further loss of genetic variation that is detrimental to the long–term survival of the population. Modifications to the breeding programme could alleviate this problem.

Phosphoinositide 3-Kinase-C2α Regulates Polycystin-2 Ciliary Entry and Protects against Kidney Cyst Formation

Signaling from the primary cilium regulates kidney tubule development and cyst formation. However, the mechanism controlling targeting of ciliary components necessary for cilium morphogenesis and signaling is largely unknown. Here, we studied the function of class II phosphoinositide 3-kinase-C2α (PI3K-C2α) in renal tubule-derived inner medullary collecting duct 3 cells and show that PI3K-C2α resides at the recycling endosome compartment in proximity to the primary cilium base. In this subcellular location, PI3K-C2α controlled the activation of Rab8, a key mediator of cargo protein targeting to the primary cilium. Consistently, partial reduction of PI3K-C2α was sufficient to impair elongation of the cilium and the ciliary transport of polycystin-2, as well as to alter proliferation signals linked to polycystin activity. In agreement, heterozygous deletion of PI3K-C2α in mice induced cilium elongation defects in kidney tubules and predisposed animals to cyst development, either in genetic models of polycystin-1/2 reduction or in response to ischemia/reperfusion-induced renal damage. These results indicate that PI3K-C2α is required for the transport of ciliary components such as polycystin-2, and partial loss of this enzyme is sufficient to exacerbate the pathogenesis of cystic kidney disease.

Giant Galápagos tortoises; molecular genetic analyses identify a trans-island hybrid in a repatriation program of an endangered taxon

BackgroundGiant Galápagos tortoises on the island of Española have been the focus of an intensive captive breeding-repatriation programme for over 35 years that saved the taxon from extinction. However, analysis of 118 samples from released individuals indicated that the bias sex ratio and large variance in reproductive success among the 15 breeders has severely reduced the effective population size (Ne).ResultsWe report here that an analysis of an additional 473 captive-bred tortoises released back to the island reveals an individual (E1465) that exhibits nuclear microsatellite alleles not found in any of the 15 breeders. Statistical analyses incorporating genotypes of 304 field-sampled individuals from all populations on the major islands indicate that E1465 is most probably a hybrid between an Española female tortoise and a male from the island of Pinzón, likely present on Española due to human transport.ConclusionRemoval of E1465 as well as its father and possible (half-)siblings is warranted to prevent further contamination within this taxon of particular conservation significance. Despite this detected single contamination, it is highly noteworthy to emphasize the success of this repatriation program conducted over nearly 40 years and involving release of over 2000 captive-bred tortoises that now reproduce in situ. The incorporation of molecular genetic analysis of the program is providing guidance that will aid in monitoring the genetic integrity of this ambitious effort to restore a unique linage of a spectacular animal.

A Single-Amino-Acid Substitution in Obg Activates a New Programmed Cell Death Pathway in Escherichia coli

ABSTRACT Programmed cell death (PCD) is an important hallmark of multicellular organisms. Cells self-destruct through a regulated series of events for the benefit of the organism as a whole. The existence of PCD in bacteria has long been controversial due to the widely held belief that only multicellular organisms would profit from this kind of altruistic behavior at the cellular level. However, over the past decade, compelling experimental evidence has established the existence of such pathways in bacteria. Here, we report that expression of a mutant isoform of the essential GTPase ObgE causes rapid loss of viability in Escherichia coli. The physiological changes that occur upon expression of this mutant protein—including loss of membrane potential, chromosome condensation and fragmentation, exposure of phosphatidylserine on the cell surface, and membrane blebbing—point to a PCD mechanism. Importantly, key regulators and executioners of known bacterial PCD pathways were shown not to influence this cell death program. Collectively, our results suggest that the cell death pathway described in this work constitutes a new mode of bacterial PCD. IMPORTANCE Programmed cell death (PCD) is a well-known phenomenon in higher eukaryotes. In these organisms, PCD is essential for embryonic development—for example, the disappearance of the interdigital web—and also functions in tissue homeostasis and elimination of pathogen-invaded cells. The existence of PCD mechanisms in unicellular organisms like bacteria, on the other hand, has only recently begun to be recognized. We here demonstrate the existence of a bacterial PCD pathway that induces characteristics that are strikingly reminiscent of eukaryotic apoptosis, such as fragmentation of DNA, exposure of phosphatidylserine on the cell surface, and membrane blebbing. Our results can provide more insight into the mechanism and evolution of PCD pathways in higher eukaryotes. More importantly, especially in the light of the looming antibiotic crisis, they may point to a bacterial Achilles’ heel and can inspire innovative ways of combating bacterial infections, directed at the targeted activation of PCD pathways. Programmed cell death (PCD) is a well-known phenomenon in higher eukaryotes. In these organisms, PCD is essential for embryonic development—for example, the disappearance of the interdigital web—and also functions in tissue homeostasis and elimination of pathogen-invaded cells. The existence of PCD mechanisms in unicellular organisms like bacteria, on the other hand, has only recently begun to be recognized. We here demonstrate the existence of a bacterial PCD pathway that induces characteristics that are strikingly reminiscent of eukaryotic apoptosis, such as fragmentation of DNA, exposure of phosphatidylserine on the cell surface, and membrane blebbing. Our results can provide more insight into the mechanism and evolution of PCD pathways in higher eukaryotes. More importantly, especially in the light of the looming antibiotic crisis, they may point to a bacterial Achilles’ heel and can inspire innovative ways of combating bacterial infections, directed at the targeted activation of PCD pathways.

Apolipoproteins L control cell death triggered by TLR3/TRIF signaling in dendritic cells.

Apolipoproteins L (ApoLs) are Bcl‐2‐like proteins expressed under inflammatory conditions in myeloid and endothelial cells. We found that Toll‐like receptor (TLR) stimuli, particularly the viral mimetic polyinosinic:polycytidylic acid (poly(I:C)), specifically induce ApoLs7/11 subfamilies in murine CD8α+ dendritic cells (DCs). This induction requires the TLR3/TRIF (where TRIF is TIR domain containing adapter‐inducing interferon β) signaling pathway and is dependent on IFN‐β in all ApoLs subfamilies except for ApoL7c. Poly(I:C) treatment of DCs is also associated with induction of both cell death and autophagy. ApoLs expression is related to promotion of DC death by poly(I:C), as ApoLs7/11 knockdown increases DC survival and ApoLs7 are associated with the anti‐apoptotic protein Bcl‐xL (where Bcl‐xL is B‐cell lymphoma extra large). Similarly, in human monocyte‐derived DCs poly(I:C) induces both cell death and the expression of ApoLs, principally ApoL3. Finally, the BH3‐like peptide of ApoLs appears to be involved in the DC death‐promoting activity. We would like to propose that ApoLs are involved in cell death linked to activation of DCs by viral stimuli.

Development of 10 highly‐polymorphic microsatellite markers in the vulnerable Galápagos land iguanas (genus Conolophus)

The two species of Galápagos land iguanas (Conolophus subcristatus and C. pallidus) are listed as ‘vulnerable’ species by the International Union for the Conservation of Nature (IUCN Red List; http://www.iucnredlist.org). Here, we report on the isolation and characterization of 10 microsatellite markers using 562 individuals sampled on all Galápagos islands where Conolophus species occur today. We show that these 10 loci are highly polymorphic and display diagnostic alleles for five out of the six island populations. These markers will be useful for Conolophus population genetic analyses as well as for guiding ongoing captive breeding programmes.

A Mutant Isoform of ObgE Causes Cell Death by Interfering with Cell Division

Cell division is a vital part of the cell cycle that is fundamental to all life. Despite decades of intense investigation, this process is still incompletely understood. Previously, the essential GTPase ObgE, which plays a role in a myriad of basic cellular processes (such as initiation of DNA replication, chromosome segregation, and ribosome assembly), was proposed to act as a cell cycle checkpoint in Escherichia coli by licensing chromosome segregation. We here describe the effect of a mutant isoform of ObgE (ObgE∗) that causes cell death by irreversible arrest of the cell cycle at the stage of cell division. Notably, chromosome segregation is allowed to proceed normally in the presence of ObgE∗, after which cell division is blocked. Under conditions of rapid growth, ongoing cell cycles are completed before cell cycle arrest by ObgE∗ becomes effective. However, cell division defects caused by ObgE∗ then elicit lysis through formation of membrane blebs at aberrant division sites. Based on our results, and because ObgE was previously implicated in cell cycle regulation, we hypothesize that the mutation in ObgE∗ disrupts the normal role of ObgE in cell division. We discuss how ObgE∗ could reveal more about the intricate role of wild-type ObgE in division and cell cycle control. Moreover, since Obg is widely conserved and essential for viability, also in eukaryotes, our findings might be applicable to other organisms as well.

The inner-rod component of Shigella flexneri type 3 secretion system, MxiI, is involved in the transmission of the secretion activation signal by its interaction with MxiC

The virulence of Shigella mainly resides in the use of a Type 3 Secretion System (T3SS) to inject several proteins inside the host cell. Three categories of proteins are hierarchically secreted: (1) the needle components (MxiH and MxiI), (2) the translocator proteins which form a pore (translocon) inside the host cell membrane, and (3) the effectors interfering with the host cell signaling pathways. In the absence of host cell contact, the T3SS is maintained in an “off” state by the presence of a tip complex. We have previously identified a gatekeeper protein, MxiC, which sequesters effectors inside the bacteria probably by interacting with MxiI, the inner‐rod component. Upon cell contact and translocon insertion, a signal is most likely transmitted from the top of the needle to the base, passing through the needle and allowing effectors release. However, the molecular mechanism underlying the transmission of the activation signal through the needle is still poorly understood. In this work, we investigate the role of MxiI in the activation of the T3SS by performing a mutational study. Interestingly we have shown that mutations of a single residue in MxiI (T82) induce an mxiC‐like phenotype and prevent the interaction with MxiC. Moreover, we have shown that the L26A mutation significantly reduces T3 secretion. The L26A mutation impairs the interaction between MxiI and Spa40, a keystone component of the switch between needle assembly and translocators secretion. The L26A mutation also sequesters MxiC. All these results highlight the crucial role of MxiI in regulating the secretion and transmitting the activation signal of the T3SS.

PI3K-C2α regulates Polycystin-2 ciliary entry to prevent kidney cyst formation

Results PI3K-C2a resides at the recycling endosome compartment surrounding the primary cilium base where it controls the activation of Rab8, a key mediator of cargo protein targeting to the primary cilium. Consistently, partial reduction of PI3K-C2a is sufficient to impair elongation of the cilium both in Pik3c2a-silenced IMCD3 cells and in kidney tubules of Pik3c2a mice. Importantly, absence of PI3K-C2a impairs the Rab8-dependent transport of Polycystin-2 to cilia and produces an overactivation of proliferative pathways regulated by ciliary Polycystins, such as the mTOR and MAPK pathways. Both defects can be rescued by transfection of constitutively active Rab8. In line with defective Polycystin signaling, heterozygous deletion of PI3K-C2a in mice causes an overall deregulation of proliferative signals in response to Ischemia/Reperfusioninduced renal damage, and this condition predisposes to cyst development.