scholarly journals Rab43 regulates the sorting of a subset of membrane protein cargo through the medial Golgi

2016 ◽  
Vol 27 (11) ◽  
pp. 1834-1844 ◽  
Author(s):  
John V. Cox ◽  
Rita Kansal ◽  
Michael A. Whitt
Keyword(s):  
Anion Exchanger ◽  
Small Interfering Rna ◽  
Secretory Pathway ◽  
Minimal Effect ◽  
Anterograde Transport ◽  
Gtp Binding ◽  
Golgi Compartment ◽  
Membrane Spanning ◽  
Interfering Rna

To evaluate the role of cytoplasmic domains of membrane-spanning proteins in directing trafficking through the secretory pathway, we generated fluorescently tagged VSV G tsO45 with either the native G tail (G) or a cytoplasmic tail derived from the chicken AE1-4 anion exchanger (GAE). We previously showed that these two proteins progressed through the Golgi with distinct kinetics. To investigate the basis for the differential sorting of G and GAE, we analyzed the role of several Golgi-associated small GTP-binding proteins and found that Rab43 differentially regulated their transport through the Golgi. We show that the expression of GFP-Rab43 arrested the anterograde transport of GAEin a Rab43-positive medial Golgi compartment. GFP-Rab43 expression also inhibited the acquisition of endoH-resistant sugars and the surface delivery of GAE, as well as the surface delivery of the AE1-4 anion exchanger. In contrast, GFP-Rab43 expression did not affect the glycosylation or surface delivery of G. Unexpectedly, down-regulation of endogenous Rab43 using small interfering RNA resulted in an increase in the accumulation of GAEon the cell surface while having minimal effect on the surface levels of G. Our data demonstrate that Rab43 regulates the sorting of a subset of membrane-spanning cargo as they progress through the medial Golgi.

scholarly journals Phospholipase C γ1 regulates early secretory trafficking and cell migration via interaction with p115

2015 ◽  
Vol 26 (12) ◽  
pp. 2263-2278 ◽  
Author(s):  
Valentina Millarte ◽  
Gaelle Boncompain ◽  
Kerstin Tillmann ◽  
Franck Perez ◽  
Elizabeth Sztul ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Migration ◽  
Phospholipase C ◽  
Small Interfering Rna ◽  
Secretory Pathway ◽  
Early Secretory Pathway ◽  
Catalytic Function ◽  
Golgi Structure ◽  
Interfering Rna

The role of early secretory trafficking in the regulation of cell motility remains incompletely understood. Here we used a small interfering RNA screen to monitor the effects on structure of the Golgi apparatus and cell migration. Two major Golgi phenotypes were observed—fragmented and small Golgi. The latter exhibited a stronger correlation with a defect in cell migration. Among the small Golgi hits, we focused on phospholipase C γ1 (PLCγ1). We show that PLCγ1 regulates Golgi structure and cell migration independently of its catalytic activity but in a manner that depends on interaction with the tethering protein p115. PLCγ1 regulates the dynamics of p115 in the early secretory pathway, thereby controlling trafficking from the endoplasmic reticulum to the Golgi. Our results uncover a new function of PLCγ1 that is independent of its catalytic function and link early secretory trafficking to the regulation of cell migration.

scholarly journals Rab1b regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments.

1991 ◽  
Vol 115 (1) ◽  
pp. 31-43 ◽  
Author(s):  
H Plutner ◽  
A D Cox ◽  
S Pind ◽  
R Khosravi-Far ◽  
J R Bourne ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Essential Role ◽  
Secretory Pathway ◽  
Binding Protein ◽  
Vesicular Transport ◽  
Initial Step ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Golgi Compartment

We report an essential role for the ras-related small GTP-binding protein rab1b in vesicular transport in mammalian cells. mAbs detect rab1b in both the ER and Golgi compartments. Using an assay which reconstitutes transport between the ER and the cis-Golgi compartment, we find that rab1b is required during an initial step in export of protein from the ER. In addition, it is also required for transport of protein between successive cis- and medial-Golgi compartments. We suggest that rab1b may provide a common link between upstream and downstream components of the vesicular fission and fusion machinery functioning in early compartments of the secretory pathway.

scholarly journals Guanine Deaminase in Human Epidermal Keratinocytes Contributes to Skin Pigmentation

Molecules ◽  
2020 ◽  
Vol 25 (11) ◽  
pp. 2637
Author(s):  
Joon Min Jung ◽  
Tai Kyung Noh ◽  
Soo Youn Jo ◽  
Su Yeon Kim ◽  
Youngsup Song ◽  
...  
Keyword(s):  
Stem Cell Factor ◽  
Small Interfering Rna ◽  
Skin Pigmentation ◽  
Epidermal Keratinocytes ◽  
Melanin Content ◽  
Human Epidermal Keratinocytes ◽  
Guanine Deaminase ◽  
Keratinocyte Culture ◽  
Interfering Rna

Epidermal keratinocytes are considered as the most important neighboring cells that modify melanogenesis. Our previous study used microarray to show that guanine deaminase (GDA) gene expression is highly increased in melasma lesions. Hence, we investigated the role of GDA in skin pigmentation. We examined GDA expression in post-inflammatory hyperpigmentation (PIH) lesions, diagnosed as Riehl’s melanosis. We further investigated the possible role of keratinocyte-derived GDA in melanogenesis by quantitative PCR, immunofluorescence staining, small interfering RNA-based GDA knockdown, and adenovirus-mediated GDA overexpression. We found higher GDA positivity in the hyperpigmentary lesional epidermis than in the perilesional epidermis. Both UVB irradiation and stem cell factor (SCF) plus endothelin-1 (ET-1) were used, which are well-known melanogenic stimuli upregulating GDA expression in both keratinocyte culture alone and keratinocyte and melanocyte coculture. GDA knockdown downregulated melanin content, while GDA overexpression promoted melanogenesis in the coculture. When melanocytes were treated with UVB-exposed keratinocyte-conditioned media, the melanin content was increased. Also, GDA knockdown lowered SCF and ET-1 expression levels in keratinocytes. GDA in epidermal keratinocytes may promote melanogenesis by upregulating SCF and ET-1, suggesting its role in skin hyperpigmentary disorders.

scholarly journals Functional Analysis of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases Differentially Expressed by Variants of Human HT-1080 Fibrosarcoma Exhibiting High and Low Levels of Intravasation and Metastasis

2007 ◽  
Vol 282 (49) ◽  
pp. 35964-35977 ◽  
Author(s):  
Juneth J. Partridge ◽  
Mark A. Madsen ◽  
Veronica C. Ardi ◽  
Thales Papagiannakopoulos ◽  
Tatyana A. Kupriyanova ◽  
...  
Keyword(s):  
Matrix Metalloproteinases ◽  
Cancer Cell ◽  
Small Interfering Rna ◽  
Cultured Cells ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Down Regulation ◽  
Primary Tumors ◽  
Tissue Inhibitors ◽  
Interfering Rna

The role of tumor-derived matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) in cancer cell dissemination was analyzed by employing two variants of human HT-1080 fibrosarcoma, HT-hi/diss and HT-lo/diss, which differ by 50-100-fold in their ability to intravasate and metastasize in the chick embryo. HT-hi/diss and HT-lo/diss were compared by quantitative reverse transcription-PCR and Western blot analyses for mRNA and protein expression of nine MMPs (MMP-1, -2, -3, -7, -8, -9, -10, -13, and -14) and three TIMPs (TIMP-1, -2, and -3) in cultured cells in vitro and in primary tumors in vivo. MMP-1 and MMP-9 were more abundant in the HT-hi/diss variant, both in cultures and in tumors, whereas the HT-lo/diss variant consistently expressed higher levels of MMP-2, TIMP-1, and TIMP-2. Small interfering RNA-mediated down-regulation of MMP-2 and TIMP-2 increased intravasation of HT-lo/diss cells. Coordinately, treatment of the developing HT-hi/diss tumors with recombinant TIMP-1 and TIMP-2 significantly reduced HT-hi/diss cell intravasation. However, a substantial increase of HT-hi/diss dissemination was observed upon small interfering RNA-mediated down-regulation of three secreted MMPs, including the interstitial collagenase MMP-1 and the two gelatinases, MMP-2 and MMP-9, but not the membrane-tethered MMP-14. The addition of recombinant pro-MMP-9 protein to the HT-hi/diss tumors reversed the increased intravasation of HT-hi/diss cells, in which MMP-9 was stably down-regulated by short hairpin RNA interference. This rescue did not occur if the pro-MMP-9 was stoichiometrically complexed with TIMP-1, pointing to a direct role of the MMP-9 enzyme in regulation of HT-hi/diss intravasation. Collectively, these findings demonstrate that tumor-derived MMPs may have protective functions in cancer cell intravasation, i.e. not promoting but rather catalytically interfering with the early stages of cancer dissemination.

scholarly journals Elucidating the role of surface chemistry on cationic phosphorus dendrimer–siRNA complexation

Nanoscale ◽  
2018 ◽  
Vol 10 (23) ◽  
pp. 10952-10962 ◽  
Author(s):  
Marco A. Deriu ◽  
Nicolas Tsapis ◽  
Magali Noiray ◽  
Gianvito Grasso ◽  
Nabil El Brahmi ◽  
...  
Keyword(s):  
Surface Chemistry ◽  
Structural Properties ◽  
Crucial Role ◽  
Small Interfering Rna ◽  
Sirna Delivery ◽  
Interfering Rna

In the field of dendrimers targeting small interfering RNA (siRNA) delivery, dendrimer structural properties, such as the surface chemistry, play a crucial role in the efficiency of complexation.

scholarly journals A Role for Fis1 in Both Mitochondrial and Peroxisomal Fission in Mammalian Cells

2005 ◽  
Vol 16 (11) ◽  
pp. 5077-5086 ◽  
Author(s):  
Annett Koch ◽  
Yisang Yoon ◽  
Nina A. Bonekamp ◽  
Mark A. McNiven ◽  
Michael Schrader
Keyword(s):  
Mammalian Cells ◽  
Small Interfering Rna ◽  
Transmembrane Domain ◽  
Mitochondrial Fission ◽  
Ectopic Expression ◽  
Mammalian Homologue ◽  
Necessary And Sufficient ◽  
Interfering Rna ◽  
Peroxisome Division

The mammalian dynamin-like protein DLP1/Drp1 has been shown to mediate both mitochondrial and peroxisomal fission. In this study, we have examined whether hFis1, a mammalian homologue of yeast Fis1, which has been shown to participate in mitochondrial fission by an interaction with DLP1/Drp1, is also involved in peroxisomal growth and division. We show that hFis1 localizes to peroxisomes in addition to mitochondria. Through differential tagging and deletion experiments, we demonstrate that the transmembrane domain and the short C-terminal tail of hFis1 is both necessary and sufficient for its targeting to peroxisomes and mitochondria, whereas the N-terminal region is required for organelle fission. hFis1 promotes peroxisome division upon ectopic expression, whereas silencing of Fis1 by small interfering RNA inhibited fission and caused tubulation of peroxisomes. These findings provide the first evidence for a role of Fis1 in peroxisomal fission and suggest that the fission machinery of mitochondria and peroxisomes shares common components.

scholarly journals Cofilin-mediated sorting and export of specific cargo from the Golgi apparatus in yeast

2012 ◽  
Vol 23 (12) ◽  
pp. 2327-2338 ◽  
Author(s):  
Amy J. Curwin ◽  
Julia von Blume ◽  
Vivek Malhotra
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Calcium Atpase ◽  
Carboxypeptidase Y ◽  
Secretory Proteins ◽  
Golgi Membranes ◽  
Golgi Compartment ◽  
Reduced Rate ◽  
Golgi Network

The mechanism of cargo sorting at the trans-Golgi network (TGN) for secretion is poorly understood. We previously reported the involvement of the actin-severing protein cofilin and the Ca2+ ATPase secretory pathway calcium ATPase 1 (SPCA1) in the sorting of soluble secretory cargo at the TGN in mammalian cells. Now we report that cofilin in yeast is required for export of selective secretory cargo at the late Golgi membranes. In cofilin mutant (cof1-8) cells, the cell wall protein Bgl2 was secreted at a reduced rate and retained in a late Golgi compartment, whereas the plasma membrane H+ ATPase Pma1, which is transported in the same class of carriers, reached the cell surface. In addition, sorting of carboxypeptidase Y (CPY) to the vacuole was delayed, and CPY was secreted from cof1-8 cells. Loss of the yeast orthologue of SPCA1 (Pmr1) exhibited similar sorting defects and displayed synthetic sickness with cof1-8. In addition, overexpression of PMR1 restored Bgl2 secretion in cof1-8 cells. These findings highlight the conserved role of cofilin and SPCA1/Pmr1 in sorting of the soluble secretory proteins at the TGN/late Golgi membranes in eukaryotes.

Soluble CD40 Ligand Promotes Macrophage Foam Cell Formation in the Etiology of Atherosclerosis

Cardiology ◽  
2015 ◽  
Vol 131 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Ming Yuan ◽  
Hongjie Fu ◽  
Lifen Ren ◽  
Haichang Wang ◽  
Wenyi Guo
Keyword(s):  
Small Interfering Rna ◽  
Foam Cell ◽  
Cell Formation ◽  
Cd40 Ligand ◽  
Foam Cell Formation ◽  
Lipid Deposition ◽  
Soluble Cd40 Ligand ◽  
Important Indicator ◽  
Interfering Rna

Objective: High levels of soluble CD40 ligand (sCD40L) in the circulation have been suggested as an important indicator of cardiovascular diseases such as atherosclerosis and acute coronary syndromes. In the present study, we explored the role of sCD40L in the formation of foam cells. Methods: Lipid deposition and foam cell formation was measured by high-performance liquid chromatography and Nile Red staining, respectively. Gene expressions were detected by quantitative real-time PCR and Western blot analysis. The interaction between CD40 and sCD40L were blocked by CD40 small interfering RNA or anti-CD40 antibody. Results: sCD40L significantly increased lipid deposition and foam cell formation associated with upregulation of scavenger receptor type A and CD36. Additionally, sCD40L increased adipocyte enhancer-binding protein 1 and cholesterol efflux, and activated NF-κB in macrophages. sCD40L promoted foam cell formation via CD40 ligation and disruption of the ligation between CD40 and CD40L either by small interfering RNA or by a blocking anti-CD40 antibody apparently inhibiting foam cell formation in response to sCD40L. Conclusion: Our data suggests a novel insight into the role of sCD40L in foam cell formation during atherosclerosis, which further confirms the importance of sCD40L in atherosclerosis and as a target for the treatment of this disease.

Role of Rac1-regulated signaling in medulloblastoma invasion

2009 ◽  
Vol 4 (2) ◽  
pp. 97-104 ◽  
Author(s):  
Salvatore Zavarella ◽  
Mitsutoshi Nakada ◽  
Shawn Belverud ◽  
Salvatore J. Coniglio ◽  
Amanda Chan ◽  
...  
Keyword(s):  
Map Kinase ◽  
Cell Invasion ◽  
Small Interfering Rna ◽  
Immunohistochemical Analysis ◽  
Mitogen Activated Protein Kinase ◽  
Medulloblastoma Cell ◽  
Interfering Rna ◽  
Map Kinase Pathways

Object Medulloblastomas are the most common malignant brain tumors in children. These tumors are highly invasive, and patients harboring these lesions are frequently diagnosed with distant spread. In this study, the authors investigated the role of Rac1, a member of the Rho family of small guanosine triphosphatases, in medulloblastoma invasion. Methods Three established medulloblastoma cell lines were used: DAOY, UW-228, and ONS-76. Specific depletion of Rac1 protein was accomplished by transient transfection of small interfering RNA. Cell invasion through extracellular matrix (Matrigel) was quantified using a transwell migration assay. Mitogen activated protein kinase activation was determined using phospho-MAP kinase–specific antibodies, and inhibition of MAP kinase pathways was achieved by specific small molecule inhibitors. Localization of Rac1 and its expression levels were determined by immunohistochemical analysis using a Rac1-specific antibody, and Rac1 activation was qualitatively assessed by Rac1 plasma membrane association. Results Small interfering RNA–mediated depletion of Rac1 strongly inhibited medulloblastoma cell invasion. Although depletion of Rac1 inhibited the proliferation of UW-228 cells, and of ONS-76 cells to a lesser extent, it stimulated the proliferation of DAOY cells. Depletion of Rac1 also inhibited the activation of the ERK and JNK MAP kinase pathways, and inhibition of either pathway diminished invasion and proliferation. Immunohistochemical analysis demonstrated that the Rac1 protein was overexpressed in all medulloblastoma tumors examined, and indicated that Rac1 was hyperactive in 6 of 25 tumors. Conclusions The authors' data show that Rac1 is necessary for the invasive behavior of medulloblastoma cells in vitro, and plays a variable role in medulloblastoma cell proliferation. In addition, these results indicate that Rac1 stimulates medulloblastoma invasion by activating the ERK and JNK pathways. The authors suggest that Rac1 and signaling elements controlled by this guanosine triphosphatase may serve as novel targets for therapeutic intervention in malignant medulloblastomas.

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