scholarly journals An SNX10-dependent mechanism down-regulates fusion between mature osteoclasts

2021 ◽  
Author(s):  
Maayan Barnea Zohar ◽  
Sabina E. Winograd-Katz ◽  
Moran Shalev ◽  
Esther Arman ◽  
Nina Reuven ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Lipid Binding ◽  
Vesicular Trafficking ◽  
Wild Type ◽  
Binding Properties ◽  
Sorting Nexin ◽  
A Cell ◽  
Altered Lipid ◽  
Dependent Mechanism

Homozygosity for the R51Q mutation in sorting nexin 10 (SNX10) inactivates osteoclasts (OCLs) and induces autosomal recessive osteopetrosis in humans and in mice. We show here that fusion of wild-type murine monocytes to form OCLs is highly regulated, and that its extent is limited by blocking fusion between mature OCLs. In contrast, monocytes from homozygous R51Q SNX10 mice fuse uncontrollably, forming giant dysfunctional OCLs that can become 10-100 fold larger than their wild-type counterparts. Furthermore, mutant OCLs display reduced endocytotic activity, suggesting that their deregulated fusion is due to alterations in membrane homeostasis caused by loss of SNX10 function. This is supported by the finding that the R51Q SNX10 protein is unstable and exhibits altered lipid-binding properties, and is consistent with a key role for SNX10 in vesicular trafficking. We propose that OCL size and functionality are regulated by a cell-autonomous, SNX10-dependent mechanism that down-regulates fusion between mature OCLs. The R51Q mutation abolishes this regulatory activity, leading to excessive fusion, loss of bone resorption capacity and, consequently, to an osteopetrotic phenotype in vivo.

scholarly journals DJ-1 regulates the integrity and function of ER-mitochondria association through interaction with IP3R3-Grp75-VDAC1

2019 ◽  
Vol 116 (50) ◽  
pp. 25322-25328 ◽  
Author(s):  
Yi Liu ◽  
Xiaopin Ma ◽  
Hisashi Fujioka ◽  
Jun Liu ◽  
Shengdi Chen ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Cellular Mechanism ◽  
Loss Of Function ◽  
Wild Type ◽  
Calcium Efflux ◽  
And Function ◽  
The Brain ◽  
Early Onset Parkinson’S Disease

Loss-of-function mutations in DJ-1 are associated with autosomal recessive early onset Parkinson’s disease (PD), yet the underlying pathogenic mechanism remains elusive. Here we demonstrate that DJ-1 localized to the mitochondria-associated membrane (MAM) both in vitro and in vivo. In fact, DJ-1 physically interacts with and is an essential component of the IP3R3-Grp75-VDAC1 complexes at MAM. Loss of DJ-1 disrupted the IP3R3-Grp75-VDAC1 complex and led to reduced endoplasmic reticulum (ER)-mitochondria association and disturbed function of MAM and mitochondria in vitro. These deficits could be rescued by wild-type DJ-1 but not by the familial PD-associated L166P mutant which had demonstrated reduced interaction with IP3R3-Grp75. Furthermore, DJ-1 ablation disturbed calcium efflux-induced IP3R3 degradation after carbachol treatment and caused IP3R3 accumulation at the MAM in vitro. Importantly, similar deficits in IP3R3-Grp75-VDAC1 complexes and MAM were found in the brain of DJ-1 knockout mice in vivo. The DJ-1 level was reduced in the substantia nigra of sporadic PD patients, which was associated with reduced IP3R3-DJ-1 interaction and ER-mitochondria association. Together, these findings offer insights into the cellular mechanism in the involvement of DJ-1 in the regulation of the integrity and calcium cross-talk between ER and mitochondria and suggests that impaired ER-mitochondria association could contribute to the pathogenesis of PD.

scholarly journals Quorum Sensing: a Transcriptional Regulatory System Involved in the Pathogenicity of Burkholderia mallei

2004 ◽  
Vol 72 (11) ◽  
pp. 6589-6596 ◽  
Author(s):  
Ricky L. Ulrich ◽  
David DeShazer ◽  
Harry B. Hines ◽  
Jeffrey A. Jeddeloh
Keyword(s):  
Quorum Sensing ◽  
Virulence Factor ◽  
Regulatory System ◽  
In Silico Analysis ◽  
Homoserine Lactone ◽  
Burkholderia Mallei ◽  
Wild Type ◽  
Transcriptional Regulatory ◽  
A Cell

ABSTRACT Numerous gram-negative bacterial pathogens regulate virulence factor expression by using a cell density mechanism termed quorum sensing (QS). An in silico analysis of the Burkholderia mallei ATCC 23344 genome revealed that it encodes at least two luxI and four luxR homologues. Using mass spectrometry, we showed that wild-type B. mallei produces the signaling molecules N-octanoyl-homoserine lactone and N-decanoyl-homoserine lactone. To determine if QS is involved in the virulence of B. mallei, we generated mutations in each putative luxIR homologue and tested the pathogenicities of the derivative strains in aerosol BALB/c mouse and intraperitoneal hamster models. Disruption of the B. mallei QS alleles, especially in RJ16 (bmaII) and RJ17 (bmaI3), which are luxI mutants, significantly reduced virulence, as indicated by the survival of mice who were aerosolized with 104 CFU (10 50% lethal doses [LD50s]). For the B. mallei transcriptional regulator mutants (luxR homologues), mutation of the bmaR5 allele resulted in the most pronounced decrease in virulence, with 100% of the challenged animals surviving a dose of 10 LD50s. Using a Syrian hamster intraperitoneal model of infection, we determined the LD50s for wild-type B. mallei and each QS mutant. An increase in the relative LD50 was found for RJ16 (bmaI1) (>967 CFU), RJ17 (bmaI3) (115 CFU), and RJ20 (bmaR5) (151 CFU) compared to wild-type B. mallei (<13 CFU). These findings demonstrate that B. mallei carries multiple luxIR homologues that either directly or indirectly regulate the biosynthesis of an essential virulence factor(s) that contributes to the pathogenicity of B. mallei in vivo.

scholarly journals R51Q SNX10 induces osteopetrosis by promoting uncontrolled fusion of monocytes to form giant, non-functional osteoclasts

2018 ◽  
Author(s):  
Maayan Barnea ◽  
Merle Stein ◽  
Sabina Winograd-Katz ◽  
Moran Shalev ◽  
Esther Arman ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Mouse Model ◽  
Autosomal Recessive ◽  
Molecular Mechanisms ◽  
Precursor Cell ◽  
Unique Combination ◽  
Sorting Nexin ◽  
Autosomal Recessive Osteopetrosis

SummaryThe molecular mechanisms that regulate fusion of monocytes into functional osteoclasts are virtually unknown. We describe a knock-in mouse model for the R51Q mutation in sorting nexin 10 (SNX10) that exhibits osteopetrosis and related symptoms of patients of autosomal recessive osteopetrosis linked to this mutation. Osteopetrosis arises in homozygous R51Q SNX10 mice due to a unique combination of reduced numbers of osteoclasts that are non-functional. Fusion of mutant monocytes is deregulated and occurs rapidly and continuously to form giant, non-functional osteoclasts. Mutant osteoclasts mature quickly and survive poorly in vitro, possibly accounting for their scarcity in vivo. These cells also exhibit impaired ruffled borders, which are required for bone resorption, providing an additional basis for the osteopetrotic phenotype. More broadly, we propose that the maximal size of osteoclasts is actively determined by a genetically-regulated, cell-autonomous mechanism that limits precursor cell fusion, and for which SNX10 is required.

scholarly journals Regulation of cilia abundance in multiciliated cells

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Rashmi Nanjundappa ◽  
Dong Kong ◽  
Kyuhwan Shim ◽  
Tim Stearns ◽  
Steven L Brody ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Surface Area ◽  
Ex Vivo ◽  
Compensatory Mechanism ◽  
Multiciliated Cells ◽  
Culture Model ◽  
A Cell ◽  
Dependent Mechanism

Multiciliated cells (MCC) contain hundreds of motile cilia used to propel fluid over their surface. To template these cilia, each MCC produces between 100-600 centrioles by a process termed centriole amplification. Yet, how MCC regulate the precise number of centrioles and cilia remains unknown. Airway progenitor cells contain two parental centrioles (PC) and form structures called deuterosomes that nucleate centrioles during amplification. Using an ex vivo airway culture model, we show that ablation of PC does not perturb deuterosome formation and centriole amplification. In contrast, loss of PC caused an increase in deuterosome and centriole abundance, highlighting the presence of a compensatory mechanism. Quantification of centriole abundance in vitro and in vivo identified a linear relationship between surface area and centriole number. By manipulating cell size, we discovered that centriole number scales with surface area. Our results demonstrate that a cell-intrinsic surface area-dependent mechanism controls centriole and cilia abundance in multiciliated cells.

Different Pathways of Cell Killing by Gossypol Enantiomers

2002 ◽  
Vol 227 (6) ◽  
pp. 398-401 ◽  
Author(s):  
Jianping Qiu ◽  
Lonny R. Levin ◽  
Jochen Buck ◽  
Marcus M. Reidenberg
Keyword(s):  
Cell Line ◽  
Tumor Size ◽  
Side Effect ◽  
Side Effect Profile ◽  
Wild Type ◽  
Specific Mechanism ◽  
Therapeutic Drugs ◽  
A Cell ◽  
Selective Effects

Gossypol, a polyphenolic, aldehyde-containing constituent of cottonseed, produced partial responses (>50% reduction in tumor size) in some patients with advanced cancer and suppressed sperm as an antifertility agent for men. This action in vivo and its novel side effect profile suggest a specific mechanism of the action of gossypol. Using the random homozygous knockout approach of Li and Cohen (1), we developed a cell line resistant to killing by gossypol, but sensitive to methotrexate and doxorubicin. It showed stereospecific resistance to killing by (–) gossypol (ED50 4.9 μM) compared with wild type (ED50 2.0 μM). The resistant and wild-type cells were equally sensitive to (+) gossypol (ED50 8.8 and 8.4 μM, respectively), methotrexate, and doxyrubicin. We conclude that gossypol affects cells by a stereospecific pathway for (–) gossypol, possibly related to its selective effects, and a nonstereospecific pathway for (+) gossypol and higher concentrations of (-) gossypol. Further knowledge about the stereospecific pathway may lead to new therapeutic drugs.

scholarly journals Recombinant Listeria monocytogenes Expressing a Cell Wall-Associated Listeriolysin O Is Weakly Virulent but Immunogenic

2009 ◽  
Vol 77 (10) ◽  
pp. 4371-4382 ◽  
Author(s):  
Javier A. Carrero ◽  
Boris Calderon ◽  
Hector Vivanco-Cid ◽  
Emil R. Unanue
Keyword(s):  
Listeria Monocytogenes ◽  
Listeriolysin O ◽  
Wild Type ◽  
Positive Bacterium ◽  
Cell Responses ◽  
A Cell ◽  
Weakly Virulent

ABSTRACT Listeriolysin O (LLO) is an essential virulence factor for the gram-positive bacterium Listeria monocytogenes. Our goal was to determine if altering the topology of LLO would alter the virulence and toxicity of L. monocytogenes in vivo. A recombinant strain was generated that expressed a surface-associated LLO (sLLO) variant secreted at 40-fold-lower levels than the wild type. In culture, the sLLO strain grew in macrophages, translocated to the cytosol, and induced cell death. However, the sLLO strain showed decreased infectivity, reduced lymphocyte apoptosis, and decreased virulence despite a normal in vitro phenotype. Thus, the topology of LLO in L. monocytogenes was a factor in the pathogenesis of the infection and points to a role of LLO secretion during in vivo infection. The sLLO strain was cleared by severe combined immunodeficient (SCID) mice. Despite the attenuation of virulence, the sLLO strain was immunogenic and capable of eliciting protective T-cell responses.

scholarly journals The Vaccinia Virus G1L Putative Metalloproteinase Is Essential for Viral Replication In Vivo

2004 ◽  
Vol 78 (18) ◽  
pp. 9947-9953 ◽  
Author(s):  
Marika Hedengren-Olcott ◽  
Chelsea M. Byrd ◽  
Jeffrey Watson ◽  
Dennis E. Hruby
Keyword(s):  
Vaccinia Virus ◽  
Structural Similarity ◽  
Wild Type ◽  
Similar Time ◽  
Α Subunit ◽  
Mitochondrial Processing Peptidase ◽  
Core Proteins ◽  
A Cell ◽  
Viral Maturation

ABSTRACT The function of the putative metalloproteinase encoded by the vaccinia virus G1L gene is unknown. To address this question, we have generated a vaccinia virus strain in which expression of the G1L gene is dependent on the addition of tetracycline (TET) when infection proceeds in a cell line expressing the tetracycline repressor. The vvtetOG1L virus replicated similarly to wild-type Western Reserve (WR) virus in these cells when TET was present but was arrested at a late stage in viral maturation in the absence of TET. This arrest resulted in the accumulation of 98.5% round immature virus particles compared to 6.9% at a similar time point when TET was present. Likewise, the titer of infectious virus progeny decreased by 98.9% ± 0.97% when the vvtetOG1L virus was propagated in the absence of TET. Mutant virus replication was partially rescued by plasmid-encoded G1L, but not by G1L containing an HXXEH motif mutated to RXXQR. Modeling of G1L revealed a predicted structural similarity to the α-subunit of Saccharomyces cerevisiae mitochondrial processing peptidase (α-MPP). The HXXEH motif of G1L perfectly overlaps the HXXDR motif of α-MPP in this model. These results demonstrate that G1L is essential for virus maturation and suggest that G1L is a metalloproteinase with structural homology to α-MPP. However, no obvious effects on the expression and processing of the vaccinia virus major core proteins were observed in the G1L conditional mutant in the absence of TET compared to results for the TET and wild-type WR controls, suggesting that G1L activity is required after this step in viral morphogenesis.

scholarly journals Compartment-Restricted Biotinylation Reveals Novel Features of Prion Protein Metabolism in Vivo

2010 ◽  
Vol 21 (24) ◽  
pp. 4325-4337 ◽  
Author(s):  
Amy B. Emerman ◽  
Zai-Rong Zhang ◽  
Oishee Chakrabarti ◽  
Ramanujan S. Hegde
Keyword(s):  
Cell Surface ◽  
Prion Protein ◽  
Protein Metabolism ◽  
Selective Detection ◽  
Wild Type ◽  
Selective Labeling ◽  
A Cell ◽  
Cellular Compartments ◽  
Made In

Proteins are often made in more than one form, with alternate versions sometimes residing in different cellular compartments than the primary species. The mammalian prion protein (PrP), a cell surface GPI-anchored protein, is a particularly noteworthy example for which minor cytosolic and transmembrane forms have been implicated in disease pathogenesis. To study these minor species, we used a selective labeling strategy in which spatially restricted expression of a biotinylating enzyme was combined with asymmetric engineering of the cognate acceptor sequence into PrP. Using this method, we could show that even wild-type PrP generates small amounts of the CtmPrP transmembrane form. Selective detection of CtmPrP allowed us to reveal its N-terminal processing, long half-life, residence in both intracellular and cell surface locations, and eventual degradation in the lysosome. Surprisingly, some human disease-causing mutants in PrP selectively stabilized CtmPrP, revealing a previously unanticipated mechanism of CtmPrP up-regulation that may contribute to disease. Thus, spatiotemporal tagging has uncovered novel aspects of normal and mutant PrP metabolism and should be readily applicable to the analysis of minor topologic isoforms of other proteins.

scholarly journals Snazarus and its human ortholog SNX25 modulate autophagic flux

2021 ◽  
Author(s):  
Annie Lauzier ◽  
Marie-France Bossanyi ◽  
Raphaëlle Larcher ◽  
Sonya Nassari ◽  
Rupali Ugrankar ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Fat Body ◽  
Cellular Mechanism ◽  
Vesicular Trafficking ◽  
Autophagic Flux ◽  
Large Array ◽  
Sorting Nexin ◽  
Human Ortholog ◽  
Differential Isoform Expression

Macroautophagy, the degradation and recycling of cytosolic components in the lysosome, is an important cellular mechanism. It is a membrane-mediated process that is linked to vesicular trafficking events. The sorting nexin (SNX) protein family controls the sorting of a large array of cargoes, and various SNXs impact autophagy. To improve our understanding of their functions in vivo, we screened all Drosophila SNXs using inducible RNA interference in the fat body. Significantly, depletion of snazarus (snz) led to decreased autophagic flux. Interestingly, we observed altered distribution of Vamp7-positive vesicles with snz depletion, and snz's roles were conserved in human cells. SNX25, the closest human ortholog to snz, regulates both VAMP8 endocytosis and lipid metabolism. Through knockout-rescue experiments, we demonstrate that these activities are dependent on specific SNX25 domains and that the autophagic defects upon SNX25 loss can be rescued by ethanolamine addition. We also demonstrate the presence of differentially spliced forms of SNX14 and SNX25 in cancer cells. This work identifies a conserved role for snz/SNX25 as regulators of autophagic flux and reveals differential isoform expression between paralogs.

The secretory vesicle in living Paramecium is acidic

1989 ◽  
Vol 92 (2) ◽  
pp. 197-203 ◽  
Author(s):  
G.R. Busch ◽  
B.H. Satir
Keyword(s):  
Proton Transport ◽  
Secretory Vesicle ◽  
Secretory Proteins ◽  
Secretory Vesicles ◽  
Wild Type ◽  
Condensed Form ◽  
Mutant Cells ◽  
Acidic Compartments ◽  
Dependent Mechanism

In Paramecium, secretory proteins are packaged within membrane-bounded vesicles in a condensed form. This form expands when the proteins are released. We have now determined that a proton gradient is present in the secretory vesicles of living Paramecium. Acridine Orange, used as an in vivo indicator of acidic compartments, stained the secretory vesicles in both wild-type and mutant cells. Addition of the two agents that dissipate proton gradients (protonophores), namely, 2,4-dinitrophenol (DNP) and carbonylcyanide m-chlorophenylhydrazone (CCCP), eliminated this staining. Washed cells re-established their intravesicular acidity. Effects of sodium azide on vesicular acidity suggest that proton transport in these vesicles involves an ATP-dependent mechanism.

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