JCB

Cell Biology This Week

Just a brief post this week.  A recent paper (Miyamoto et al.) points to a requirement for nuclear actin polymerization during oocyte reprogramming by the transcription factor Oct4.  The authors had noticed an enrichment of nuclear actin-regulatory proteins in their analysis of proteins that enhance reprogramming in Xenopus oocytes.  To examine the role of actin further, they transplanted mouse nuclei into Xenopus oocytes and, using a probe to visualize filamentous actin, found that there was a large amount of actin polymerization in the nuclei.  Blocking actin polymerization by a variety of methods interfered with reprogramming stimulated by the pluripotency factor Oct4.  What regulates actin polymerization in the nucleus?  The authors focused on actin nucleators described to be present in the nucleus and found that Toca-1 overexpression enhanced Oct4’s activity.  Nuclear actin is known to be important in chromatin remodeling and this function might be how it affects Oct4-dependent reprogramming.

 

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Cell Biology This Week

A new study points to a function for a VEGF receptor in neurogenesis.  In the brain, neural stem cells, which are glial cells, exist in a vascularized niche in the subventricular zone (SVZ).  VEGFs have been shown to stimulate neurogenesis though their receptors and target cells have not been clarified.  Here, Calvo et al. examine the role of VEGF-C and its receptor VEGFR3 in neural stem cells in the SVZ.  First, they show that VEGFR3 is expressed by neural stem cells and the VEGF-C ligand is expressed in these and surrounding cells.  They then isolate VEGFR3+ cells and showed that they had properties of neural stem cells in vitro and that these properties were enhanced significantly in the presence of VEGF-C.  Specifically, VEGF-C stimulated the ability of these cells to divide.  The authors then went back to an in vivo setting and overexpressed VEGF-C in the adult mouse SVZ.  This promoted neurogenesis by increasing the survival of SVZ cells and cell cycle entry of neural stem cells.  These effects seem independent of angiogenesis and were dependent on the receptor VEGFR3.  Axon guidance molecules have been shown to be crucial for angiogenesis and this study shows that angiogenic molecules are important for neurogenesis.  It will be interesting to determine the events downstream of the receptor in neural stem cells to see if they are the same as those involved in vessel formation.

 

There is a lot of buzz about how obesity is linked to chronic inflammation and how both lead to insulin resistance and Type II diabetes.  Now, Wen et al. show that saturated fatty acids like palmitate that are part of a high-fat diet can activate the NLRP3 inflammasome.  The inflammasome is a multiprotein complex that causes activation of caspase-1 to promote the processing and release of inflammatory cytokines like IL-1beta.  IL-1beta interferes with normal insulin signaling thereby leading to insulin resistance.  Specific signaling events include the inactivation of the kinase AMPK by palmitate, which dysregulates autophagy and causes generation of mitochondrial reactive oxygen species to activate the inflammasome.  I found this paper interesting because it provides a somewhat direct route from the ingestion of a high-fat diet to inflammation and insulin resistance.

 

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Cell Biology This Week

Several papers in the last couple of weeks on neuronal cell biology have caught my eye.  In the first, Amato et al. show that the kinase AMPK contributes to neuronal polarization.  This paper shows that activation of AMPK blocks polarization and axon growth.  AMPK phosphorylates the kinesin Kif5, blocking its association with PI3K and thereby preventing PI3K accumulation at the axon tip, an event necessary for polarization.  The reason I thought this was interesting is because AMPK is a cellular energy sensor and the paper therefore connects the process of axon initiation with energy homeostasis.  Elucidating the upstream signaling events should shed light on this interesting connection.

Next, Uytterhoeven et al. uncover some interesting cell biology that could be occurring at the synapse by analyzing a fly mutant called Skywalker.  Skywalker encodes a GTPase activating protein (GAP) for Rab35 that seems to be involved in trafficking of synaptic vesicles to endosomes at the fly neuromuscular junction.  This route seems to be important for removing or recycling ubiquitinated synaptic proteins in the mutants.  It will be interesting to determine whether this pathway is important in a wildtype situation. 

Lastly, two papers point to a role for beta-adducin in synaptic plasticity in both flies and mice.  Adducins cap the barbed ends of actin filaments and link them to the spectrin cytoskeleton.  Pielage et al. show that at the fly neuromuscular junction, adducin promotes synapse stability and growth.   Using a combination of genetic and biochemical assays, the authors propose that by stabilizing the spectrin cytoskeleton, adducin promotes synapse stability and by capping actin filaments, it promotes synaptic growth.  In mice, Bednarek and Caroni show that beta-adducin is important for synapse remodeling by promoting both disassembly and subsequent re-assembly during conditions that stimulate learning and memory.

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Cell Biology This Week

The first cut may be the deepest but the final cut seems to be the hardest to grasp.  At least in the case of cytokinesis.  Contraction of an actomyosin ring narrows the connection between the two dividing cells to form an intercellular bridge. How this bridge is resolved is still a matter of debate.  Many mechanisms have been proposed for this last step of cytokinesis.  For example, in some organisms, vesicle fusion at the site of constriction is thought to contribute the membrane that enables cell separation.  Now, Guizetti et al. propose a new mechanism: membrane scission by ESCRT-III helical filaments.  Using GFP-labeled alpha tubulin, the authors examined microtubules at the intercellular bridge in HeLa cells.  They noticed that microtubules partially disassembled on one side of the bridge, on the same side as the midbody (the remnant of the central spindle that asymmetrically localizes to the constriction site).  This partial disassembly occurred coincident with abscission.  Using correlative live-cell and transmission electron microscopy, the authors noticed electron dense membrane ripples adjacent to the midbody (first observed in a very old JCB paper!) in the zone where the microtubules had disassembled. 

Black lines indicate electron-dense ridges at the intercellular bridge prior to abscission.  From Mullins and Biesele (1977).

Guizetti et al. show that the microtubule severing enzyme spastin is important for microtubule disassembly at the constriction site.  Furthermore, ESCRT-III, which is known to localize to the intercellular bridge and is essential for abscission, was enriched at the constriction zone (whereas actin and vesicles were not).  The electron-dense ripples at the constriction zone were absent in cells depleted of an ESCRT-III subunit.  ESCRT-III is targeted to the intercellular bridge by CEP55, a centrosomal protein, and ALIX, an ESCRT-related protein, both of which localize to the midbody at the time of abscission.  Electron tomography of the constriction zone in HeLa cells revealed cortical filaments perpendicular to the microtubule bundles.  These filaments formed single or intertwined helices across the intercellular bridge and were absent in cells depleted of the ESCRT-III complex by RNAi.  The authors proposed that these helical filaments are composed of ESCRT-III and that this complex provides the contractile force to deform the membrane.  This is in line with ESCRT-III function in other contexts.  Whether ESCRT-III is the molecular component of these helices and whether this mechanism occurs in vivo still remains to be shown.   Despite these major unresolved issues, the cutting-edge microscopy and interesting results make this paper worth checking out.

 

In brief, two other papers caught my eye this week.  In the first, Puneet et al. show that you can take a pathogenic molecule that is used to subvert the host immune system and exploit it to the potential benefit of the host.  Sepsis, which is a major health threat, occurs when the human body undergoes a tremendous inflammatory reaction in response to an infection.  The parasitic nematode, Acanthocheilonema viteae, produces a molecule called ES-62 that dampens the host innate immune system.  ES-62 downregulates signaling by TLR4 and TLR2—pattern-recognition receptors that recognize pathogenic molecules (TLR4, for example, recognizes the bacterial outer membrane protein lipopolysaccharide).  The authors show that ES-62 dampens these pathways by targeting MyD88—an adaptor of both of these pattern-recognition receptors—for degradation via the autophagosome (for more about autophagy and the immune system see review by Saitoh and Akira).  Importantly, when administered to mice before or after induction of sepsis, the molecule protected the majority of mice from sepsis-induced death.  The potential therapeutic benefits of these results could be quite promising. 

 

The second paper is about how ERK/MAP kinase controls lamellipodial protrusion during cell migration.  It was known previously that ERK was important for adhesion disassembly by phosphorylating the focal adhesion components FAK, Paxillin and MLCK.  Now, Mendoza et al. show that ERK localizes to the leading edge and phosphorylates and activates the WAVE2 regulatory complex (WAVE2 and Abi) that activates the actin nucleation complex Arp2/3.  Thus, this kinase that can respond to extracellular signals coordinates both focal adhesion disassembly and lamellipodial protrusion, two key events in cell migration.

 

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Cell Biology This Week

It never ceases to amaze me when there are new interactions discovered at the kinetochore, given how much attention is focused on it and how well-studied many of its components are.  For example, two recent papers now show that the centromeric protein CENP-C, which has been scrutinized for at least a couple of decades, connects the inner and outer regions of the kinetochore.  

Screpanti et al. show in human cells that the N-terminal region of Cenp-C binds directly and with high affinity to the Mis12 complex (which is part of the outer kinetochore KMN network of the protein complexes Mis12-Ndc80-Knl1).  Cenp-C is part of the CCAN (constitutive centromere-associated network) and is a part of centromeric chromatin.  Overexpression of the N-terminus of Cenp-C prevented the localization of the KMN network to kinetochores.  Predictably, these cells had chromosome segregation and spindle assembly checkpoint defects.  Meanwhile, Przewloka et al. show that each protein complex of the KMN network interacts with Cenp-C in flies, which lack many of the other proteins of the CCAN.  These authors targeted the N-terminus of Cenp-C to centrosomes and this recruited the other complexes of the KMN network and of course caused segregation defects.  So it seems that Cenp-C is the conserved connector between the inner and outer kinetochore. 

 

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Cell Biology This Week

Recently, Overholtzer et al. described a new kind of cell process called ‘entosis’ where cells engulf other cells.  Distinct from phagocytosis since the engulfed cell is not dying, entosis has been observed in both cultured cancer cell lines and tumors that are unattached to the extracellular matrix.  Now, Krajcovic et al. report that entosis can contribute to aneuploidy.  These authors noticed that in tumor cells and cultured cells bearing these cell-in-cell structures, the host cell (which has engulfed the other cell) is frequently multinucleate.  Taking a closer look, the authors imaged unattached cells in tumors and in culture undergoing division and noticed that the host cell displayed a high frequency of cytokinesis failure.  This failure occurred when the engulfed cell blocked the division plane of the host cell and resulted in the formation of binucleate cells that, upon further cell divisions, become aneuploid.  The authors call this a ‘non-genetic’ mechanism to generate aneuploidy since no mutation was induced (though clearly mutations may exist in the genomes of the tumor cells).  Skepticism aside, entotic cells could be a good model to study the role of aneuploidy in cancer since cellular control points (like the spindle assembly checkpoint) should theoretically be functioning normally and no experimental mutations have been induced. 

 

DeGennaro et al. identify a new gene, jafrac1, involved in germ cell adhesion in the fly Drosophila melanogaster.  In this organism, germ cells are set aside early in development and migrate as a group through the developing midgut to the posterior of the embryo.  Germ cells lacking jafrac1, which encodes a peroxiredoxin, show defects in adhesion to the midgut during gastrulation and as a result get excluded from the midgut.  Peroxiredoxins are enzymes that act as antioxidants by degrading hydrogen peroxide.  In line with this enzymatic function, jafrac1 mutants were more sensitive to oxidative stress.  This group had shown previously in the JCB that dynamic regulation of DE-cadherin and cell adhesion is important for germ cell migration.

 

Germ cells (green) cluster in wildtype (left) but not DE-cadherin mutant embryos (right) © 2008 Kunwar et al.

 

 

Now DeGennaro et al. show that Jafrac1 is upstream of DE-cadherin.  The authors show that exposure to hydrogen peroxide causes a downregulation of DE-cadherin and beta-catenin, both components of adherens junctions.  So it seems that peroxiredoxin, by reducing hydrogen peroxide levels, affects the stability of adherens junctions.  I admire the fact that the authors did not simply dismiss the jafrac1 phenotype or attribute it to a housekeeping function.  Although the details of this signaling pathway are still to be defined, it demonstrates that reactive oxygen species are playing very specific signaling roles during development.

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Cell Biology This Week

The blood-brain barrier (BBB) is created by special tight junctions between the endothelial cells of the brain’s vasculature.  This barrier is meant to keep out unwanted intruders, like bacteria, from the brain.  How the barrier and its selective permeability are set up are of inherent interest.  But beyond that, how some bacteria - such as the Gram-negative Neisseria meningitides - breach this divide to infect the brain and cause meningitis is also fascinating.  How do the bacteria penetrate this otherwise efficient endothelial barrier?  It is known that the bacteria in the blood stream adhere to blood vessel walls using appendages called Type IV pili.  Here they multiply and form colonies, which manipulate signaling and the actin cytoskeleton in the endothelial cells.  Now Coureuil et al. provide new molecular insight into how this bacterium gets past the BBB.   The same group had shown previously that N. meningitidis stimulates the relocalization of intercellular junction components, producing gaps in the endothelial layer through which the bacterial can squeeze through to infect the brain.  Now, these authors show that many of the events involved in this process are dependent on the G protein-coupled receptor β2AR and downstream components like β-arrestins, both of which accumulate at the plasma membrane under the bacterial colonies.  β2AR and β-arrestins are required for the ability of the bacterium to resist the shear forces that would otherwise sweep them away in the bloodstream.  β-arrestins act by promoting the docking and activation of Src to induce the formation of actin-rich protrusions that protect the bacteria from the shear forces. In addition, Coureuil et al. show that both β2AR and β-arrestins are also required for the relocalization of junction proteins: β-arrestins interact with and recruit proteins like VE-cadherin to sites of bacterial entry and away from normal intercellular junctions.  Importantly, ligand-induced endocytosis of β2AR reduces both sustained adhesion of the bacterium and their ability to cross the barrier, a finding that could hold some therapeutic potential.

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Happy New Year everyone!!

Cell Biology This Week

Developmental biologists spend a lot of time thinking about how gene regulatory networks are wired (or re-wired) during evolution or about the ‘logic’ of these networks.  A really clear example of this is presented by Booth et al. in a recent study.  These authors examined the regulatory gene network that controls mating in three yeast species: Saccharomyces cerevisiae, Kluyveromyces lactis (a dairy yeast) and Candida albicans (a human pathogen). All three species have three cell types: a, α and a/α.  The transcriptional networks that underlie the specification of these cell types are well understood in S. cerevisiae and C. albicans but not in K. lactis.  The authors decided to examine the genes specifically expressed in the haploid a and α K.lactis cells when compared to the diploid a/α cells. 

Of the haploid-specific genes that they identified, four were also haploid-specific in the other two species and had conserved roles in the mating process.  These genes are repressed by the transcription regulator a1-α2 in S. cerevisiae and C. albicans but not in K. lactis despite the presence of the a1-α2 DNA-binding sequence in other genes in K. lactis.  What is the reason behind this shift in regulation in such a conserved process?  The authors examined the upstream regulatory sequences in the promoters of the K. lactis haploid-specific genes.  A prominent motif they found was the binding sequence for the transcriptional regulator Rme1.  Rme1 is itself a haploid-specific gene in K. lactis and is regulated by a1-α2 in both S. cerevisiae and K. lactis.  It functions in processes related to mating (like meiosis and sporulation in S. cerevisiae and mating type interconversion in K. lactis).  A K. lactis Rme1 knockout displays sporulation defects so its function seems conserved. 

Does Rme1 regulate the four conserved haploid-specific genes in K. lactis?  Booth et al. show that this is indeed the case.  ChIP experiments revealed that Rme1 binds to the promoters of the four core haploid-specific genes.  Unlike their S. cerevisiae and C. albicans counterparts, K. lactis Rme1 knockout a cells were not able to form mating projections when exposed to the α pheromone.  What is the basis for this difference?  An interesting variation in the requirements for mating between the species is that K. lactis requires a starvation signal to mate.  Booth et al. show that RME1 is highly upregulated in response to starvation.  So does this gene regulate mating in K. lactis?  The authors overexpressed Rme1 in K. lactis a cells in non-starvation conditions and indeed, the a cells produced mating projections in response to the α pheromone.   So it seems that in this species of yeast, the control of mating was taken over by Rme1, a regulator that can respond to a nutritional signal.  Rme1 itself is regulated by the conserved a1-α2 heterodimer. 

I really liked this paper.  I found it to be a clear and elegant example of the re-wiring of a conserved regulatory cascade to introduce a new level of regulation.  The authors have a nice discussion of how and when (from an evolution standpoint) this may have occurred and also compare it to other situations in the development of more complex organisms.

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Cell Biology This Week

After decades of being deemed dreadfully boring by many researchers, scientists are mad about metabolism these days.  Why the change of heart?  For a start, the realization that metabolic pathways are not just in the background performing routine housekeeping functions  but are actively modulated by signaling pathways.  In fact, oncogenes stimulate aerobic glycolysis for ATP production in cancer cells (for more information about metabolic requirements of cancer cells, see this interview with one of the leaders in the field, Craig Thompson). 

Fang et al. identify a new enzyme that contributes to cancer cell metabolism.  The authors found that in PTEN-deficient cells, in which AKT is activated (AKT is part of the PI3K signaling pathway downstream of growth factor receptor signaling) there is an increase in expression of ER UDPase ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5).  This enzyme catalyzes N-glycosylation in the ER and in the process converts UDP to UMP.  Through a couple of other metabolic enzymes, this increase in UMP causes ATP conversion to AMP.  This decrease in ATP stimulates glycolysis.  PTEN is frequently lost in cancers and the authors see a correlation between PTEN loss, AKT activation and ENTPD5 upregulation in human cancer cell lines and primary tumors.  They propose that targeting this enzyme may be useful in anti-cancer therapies.  They even show that reducing expression of this enzyme in implanted tumors attenuated their growth.

Three papers in Nature indicate that hematopoietic stem cells have unique metabolic needs.  All three studies examine mouse models lacking the tumor suppressor, Lkb1, which activates AMPK, a key regulator of cellular ATP levels.  In the absence of Lkb1, Nakada et al., Gurumurthy et al. and Gan et al. all observed a depletion of hematopoietic stem and multipotent progenitor cells and eventually a depletion of all blood cell types. Lkb1 deficiency also led to increased apoptosis in these cell populations.  The cause of the defects seems to be a decrease in mitochondria function as well as other AMPK-dependent and -independent effects.   Since loss of Lkb1 seems to affect entry of hematopoietic stem cells into the cell cycle, Gurumurthy et al. propose that Lkb1 is part of a metabolic checkpoint.

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Cell Biology This Week

Big news in the stem cell field is the identification of a small molecule enhancer of hematopoietic stem cell expansion.  Boitano et al. screened 10,000 compounds to identify small molecules that promote the expansion of human primary hematopoietic stem cells (positive for cell surface marker CD34) ex vivo.  Through this approach, they identified a purine derivative that they termed StemRegenin (SR1).  In combination with a medley of cytokines, SR1 treatment improved both short- and long-term engraftment of human CD34-positive hematopoietic stem cells in immunodeficient mice.  The authors go on to identify the target of SR1—the aryl hydrocarbon receptor.   This receptor responds to certain extracellular molecules and then forms a dimer with HIF1-B. Together, these proteins activate gene expression.  The authors show that SR1 acts as an antagonist of the aryl hydrocarbon receptor.  The medical implications of their findings could be tremendous.  Furthermore, this study identifies a new pathway that inhibits stem cell differentiation.  Overall, this is a very nice example of how small molecule chemistry can uncover interesting biology.

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