The Role of Prominin-1 in the Architecture and Dynamics of Microvilli and Primary Cilia

Prominin-1 is a lipid raft–associated, cholesterol-binding membrane glycoprotein selectively associated with plasma membrane protrusions and extracellular vesicles derived therefrom. Despite its worldwide use for stem cell isolation and its clinical importance in cancer-initiating cells and photoreceptor morphogenesis the function of prominin-1 remains elusive. This prompted me to investigate its role in the architecture and dynamics of microvilli and primary cilia at the apical plasma membrane of Madin-Darby canine kidney (MDCK) cells. Therefore, stably transfected cell lines were established expressing human prominin-1 splice variant 1 or 2. Upon the overexpression of prominin-1 the number of individual microvilli and clusters of them increased significantly. I also noticed alterations in their architecture, i.e. branching microvilli. Fascinatingly, two point mutations (Pro37→Ala and Tyr41→Ser) in the ganglioside GM1-binding motif of prominin-1 increased the number of branched microvilli and generated irregular ones with knob-like structures at their tip. Additionally, the release of prominin-1+ vesicles was impaired. Interestingly, both phenotypes were suppressed by the inhibition of the phosphoinositide 3-kinase (PI3K) or the Arp2/3 complex. Impaired interaction of prominin-1 with the PI3K through the introduction of an additional mutation (Tyr828→Phe) in its PI3K-binding site also reduced the amount of structurally altered microvilli. Thus, the interaction of prominin-1 with the PI3K may drive the conversion of the docking phospholipid phosphatidylinositol(4,5)-bisphosphate into phosphatidylinositol(3,4,5)-trisphosphate resulting in the uncoupling of the microvillar membrane from the underlying actin filaments thereby creating irregular/knob-like microvilli. Simultaneously, the phospholipid conversion might modulate the activity of regulators and/or activators of the Arp2/3 complex leading to the branching of microvilli. The overexpression of human prominin-1 also increased the length of primary cilia. Remarkably, a mutation in the histone deacetylase 6-binding site that mimics acetylation produces shorter cilia in cells expressing human prominin-1.s2. Additionally, it stimulates membrane vesicle release and dome formation. Above these striking observations, I observed branching cilia and cilia with a pearling shape. Collectively, the data suggest that a complex interplay of prominin-1 with its lipid and protein interaction partners regulates the architecture and dynamics of cellular protrusions. Finally, a growing number of studies use canine prominin-1 as an antigenic marker despite the absence of specific antibodies. Studies investigating its expression in dog tissues or cells derived therefrom rely on antibodies directed against its human and murine orthologs. To determine its cross-species immunoreactivity I cloned canine prominin-1 and overexpressed it as a green fluorescent protein fusion protein in MDCK cells. Here, I show that the genomic structure of the canine prom1 gene is similar to that of human and mouse. Canine prominin-1 shows the common characteristics of the prominin-1 family but the primary structure is poorly conserved. Like human and mouse protein, it is targeted to the apical membrane of MDCK cells and specifically enriched in microvilli and primary cilia. Immunocytochemistry, flow cytometry and immunoblotting techniques revealed that none of the applied antibodies against human or mouse prominin-1 recognizes the canine protein

The Role of Prominin-1 in the Architecture and Dynamics of Microvilli and Primary Cilia

Prominin-1 is a lipid raft–associated, cholesterol-binding membrane glycoprotein selectively associated with plasma membrane protrusions and extracellular vesicles derived therefrom. Despite its worldwide use for stem cell isolation and its clinical importance in cancer-initiating cells and photoreceptor morphogenesis the function of prominin-1 remains elusive. This prompted me to investigate its role in the architecture and dynamics of microvilli and primary cilia at the apical plasma membrane of Madin-Darby canine kidney (MDCK) cells. Therefore, stably transfected cell lines were established expressing human prominin-1 splice variant 1 or 2. Upon the overexpression of prominin-1 the number of individual microvilli and clusters of them increased significantly. I also noticed alterations in their architecture, i.e. branching microvilli. Fascinatingly, two point mutations (Pro37→Ala and Tyr41→Ser) in the ganglioside GM1-binding motif of prominin-1 increased the number of branched microvilli and generated irregular ones with knob-like structures at their tip. Additionally, the release of prominin-1+ vesicles was impaired. Interestingly, both phenotypes were suppressed by the inhibition of the phosphoinositide 3-kinase (PI3K) or the Arp2/3 complex. Impaired interaction of prominin-1 with the PI3K through the introduction of an additional mutation (Tyr828→Phe) in its PI3K-binding site also reduced the amount of structurally altered microvilli. Thus, the interaction of prominin-1 with the PI3K may drive the conversion of the docking phospholipid phosphatidylinositol(4,5)-bisphosphate into phosphatidylinositol(3,4,5)-trisphosphate resulting in the uncoupling of the microvillar membrane from the underlying actin filaments thereby creating irregular/knob-like microvilli. Simultaneously, the phospholipid conversion might modulate the activity of regulators and/or activators of the Arp2/3 complex leading to the branching of microvilli. The overexpression of human prominin-1 also increased the length of primary cilia. Remarkably, a mutation in the histone deacetylase 6-binding site that mimics acetylation produces shorter cilia in cells expressing human prominin-1.s2. Additionally, it stimulates membrane vesicle release and dome formation. Above these striking observations, I observed branching cilia and cilia with a pearling shape. Collectively, the data suggest that a complex interplay of prominin-1 with its lipid and protein interaction partners regulates the architecture and dynamics of cellular protrusions. Finally, a growing number of studies use canine prominin-1 as an antigenic marker despite the absence of specific antibodies. Studies investigating its expression in dog tissues or cells derived therefrom rely on antibodies directed against its human and murine orthologs. To determine its cross-species immunoreactivity I cloned canine prominin-1 and overexpressed it as a green fluorescent protein fusion protein in MDCK cells. Here, I show that the genomic structure of the canine prom1 gene is similar to that of human and mouse. Canine prominin-1 shows the common characteristics of the prominin-1 family but the primary structure is poorly conserved. Like human and mouse protein, it is targeted to the apical membrane of MDCK cells and specifically enriched in microvilli and primary cilia. Immunocytochemistry, flow cytometry and immunoblotting techniques revealed that none of the applied antibodies against human or mouse prominin-1 recognizes the canine protein

The Role of Prominin-1 in the Architecture and Dynamics of Microvilli and Primary Cilia

Prominin-1 is a lipid raft–associated, cholesterol-binding membrane glycoprotein selectively associated with plasma membrane protrusions and extracellular vesicles derived therefrom. Despite its worldwide use for stem cell isolation and its clinical importance in cancer-initiating cells and photoreceptor morphogenesis the function of prominin-1 remains elusive. This prompted me to investigate its role in the architecture and dynamics of microvilli and primary cilia at the apical plasma membrane of Madin-Darby canine kidney (MDCK) cells. Therefore, stably transfected cell lines were established expressing human prominin-1 splice variant 1 or 2. Upon the overexpression of prominin-1 the number of individual microvilli and clusters of them increased significantly. I also noticed alterations in their architecture, i.e. branching microvilli. Fascinatingly, two point mutations (Pro37→Ala and Tyr41→Ser) in the ganglioside GM1-binding motif of prominin-1 increased the number of branched microvilli and generated irregular ones with knob-like structures at their tip. Additionally, the release of prominin-1+ vesicles was impaired. Interestingly, both phenotypes were suppressed by the inhibition of the phosphoinositide 3-kinase (PI3K) or the Arp2/3 complex. Impaired interaction of prominin-1 with the PI3K through the introduction of an additional mutation (Tyr828→Phe) in its PI3K-binding site also reduced the amount of structurally altered microvilli. Thus, the interaction of prominin-1 with the PI3K may drive the conversion of the docking phospholipid phosphatidylinositol(4,5)-bisphosphate into phosphatidylinositol(3,4,5)-trisphosphate resulting in the uncoupling of the microvillar membrane from the underlying actin filaments thereby creating irregular/knob-like microvilli. Simultaneously, the phospholipid conversion might modulate the activity of regulators and/or activators of the Arp2/3 complex leading to the branching of microvilli. The overexpression of human prominin-1 also increased the length of primary cilia. Remarkably, a mutation in the histone deacetylase 6-binding site that mimics acetylation produces shorter cilia in cells expressing human prominin-1.s2. Additionally, it stimulates membrane vesicle release and dome formation. Above these striking observations, I observed branching cilia and cilia with a pearling shape. Collectively, the data suggest that a complex interplay of prominin-1 with its lipid and protein interaction partners regulates the architecture and dynamics of cellular protrusions. Finally, a growing number of studies use canine prominin-1 as an antigenic marker despite the absence of specific antibodies. Studies investigating its expression in dog tissues or cells derived therefrom rely on antibodies directed against its human and murine orthologs. To determine its cross-species immunoreactivity I cloned canine prominin-1 and overexpressed it as a green fluorescent protein fusion protein in MDCK cells. Here, I show that the genomic structure of the canine prom1 gene is similar to that of human and mouse. Canine prominin-1 shows the common characteristics of the prominin-1 family but the primary structure is poorly conserved. Like human and mouse protein, it is targeted to the apical membrane of MDCK cells and specifically enriched in microvilli and primary cilia. Immunocytochemistry, flow cytometry and immunoblotting techniques revealed that none of the applied antibodies against human or mouse prominin-1 recognizes the canine protein

Identification of specific Tie2 cleavage sites and therapeutic modulation in experimental sepsis

Endothelial Tie2 signaling plays a pivotal role in vascular barrier maintenance at baseline and after injury. We previously demonstrated that a sharp drop in Tie2 expression observed across various murine models of critical illnesses is associated with increased vascular permeability and mortality. Matrix metalloprotease (MMP)−14-mediated Tie2 ectodomain shedding has recently been recognized as a possible mechanism for Tie2 downregulation in sepsis. Here, we identified the exact MMP14-mediated Tie2 ectodomain cleavage sites and could show that pharmacological MMP14 blockade in experimental murine sepsis exerts barrier protective and anti-inflammatory effects predominantly through the attenuation of Tie2 cleavage to improve survival both in a pre-treatment and rescue approach. Overall, we show that protecting Tie2 shedding might offer a new therapeutic opportunity for the treatment of septic vascular leakage

Dual Pharmacological Inhibition of Angiopoietin-2 and VEGF-A in Murine Experimental Sepsis

BACKGROUND Sepsis is a pathological host response to infection leading to vascular barrier breakdown due to elevated levels of angiopoietin-2 (Angpt-2) and vascular endothelial growth factor-A (VEGF-A). Here, we tested a novel heterodimeric bispecific monoclonal IgG1-cross antibody of Angpt-2 and VEGF - termed "A2V." METHODS Cecal ligation and puncture was used to induce murine polymicrobial sepsis. Organs and blood were harvested for fluorescence immunohistochemistry and RT-PCR, and survival was recorded. In vitro endothelial cells were stimulated with plasma from septic shock patients costimulated with A2V or IgG antibody followed by immunocytochemistry and real-time transendothelial electrical resistance. RESULTS Septic mice treated with A2V had a reduced induction of the endothelial adhesion molecule ICAM-1, leading to a trend towards less transmigration of inflammatory cells (A2V: 42.2 ± 1.0 vs. IgG 48.5 ± 1.7 Gr-1+ cells/HPF, p = 0.08) and reduced tissue levels of inflammatory cytokines (e.g., IL-6 mRNA: A2V 9.4 ± 3.2 vs. IgG 83.9 ± 36.7-fold over control, p = 0.03). Endothelial permeability was improved in vivo and in vitro in stimulated endothelial cells with septic plasma. Survival was improved by 38% (p = 0.02). CONCLUSION Dual inhibition of Angpt-2 and VEGF-A improves murine sepsis morbidity and mortality, making it a potential therapeutic against vascular barrier breakdown

Monoclonal Antibodies 13A4 and AC133 Do Not Recognize the Canine Ortholog of Mouse and Human Stem Cell Antigen Prominin-1 (CD133)

The pentaspan membrane glycoprotein prominin-1 (CD133) is widely used in medicine as a cell surface marker of stem and cancer stem cells. It has opened new avenues in stem cell-based regenerative therapy and oncology. This molecule is largely used with human samples or the mouse model, and consequently most biological tools including antibodies are directed against human and murine prominin-1. Although the general structure of prominin-1 including its membrane topology is conserved throughout the animal kingdom, its primary sequence is poorly conserved. Thus, it is unclear if anti-human and -mouse prominin-1 antibodies cross-react with their orthologs in other species, especially dog. Answering this issue is imperative in light of the growing number of studies using canine prominin-1 as an antigenic marker. Here, we address this issue by cloning the canine prominin-1 and use its overexpression as a green fluorescent protein fusion protein in Madin-Darby canine kidney cells to determine its immunoreactivity with antibodies against human or mouse prominin-1. We used immunocytochemistry, flow cytometry and immunoblotting techniques and surprisingly found no cross-species immunoreactivity. These results raise some caution in data interpretation when anti-prominin-1 antibodies are used in interspecies studies

Monoclonal Antibodies 13A4 and AC133 Do Not Recognize the Canine Ortholog of Mouse and Human Stem Cell Antigen Prominin-1 (CD133)

<div><p>The pentaspan membrane glycoprotein prominin-1 (CD133) is widely used in medicine as a cell surface marker of stem and cancer stem cells. It has opened new avenues in stem cell-based regenerative therapy and oncology. This molecule is largely used with human samples or the mouse model, and consequently most biological tools including antibodies are directed against human and murine prominin-1. Although the general structure of prominin-1 including its membrane topology is conserved throughout the animal kingdom, its primary sequence is poorly conserved. Thus, it is unclear if anti-human and -mouse prominin-1 antibodies cross-react with their orthologs in other species, especially dog. Answering this issue is imperative in light of the growing number of studies using canine prominin-1 as an antigenic marker. Here, we address this issue by cloning the canine prominin-1 and use its overexpression as a green fluorescent protein fusion protein in Madin-Darby canine kidney cells to determine its immunoreactivity with antibodies against human or mouse prominin-1. We used immunocytochemistry, flow cytometry and immunoblotting techniques and surprisingly found no cross-species immunoreactivity. These results raise some caution in data interpretation when anti-prominin-1 antibodies are used in interspecies studies.</p></div

Flunarizine suppresses endothelial Angiopoietin-2 in a calcium - dependent fashion in sepsis

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to an infection leading to systemic inflammation and endothelial barrier breakdown. The vascular-destabilizing factor Angiopoietin-2 (Angpt-2) has been implicated in these processes in humans. Here we screened in an unbiased approach FDA-approved compounds with respect to Angpt-2 suppression in endothelial cells (ECs) in vitro. We identified Flunarizine – a well-known anti-migraine calcium channel (CC) blocker – being able to diminish intracellular Angpt-2 protein in a time- and dose-dependent fashion thereby indirectly reducing the released protein. Moreover, Flunarizine protected ECs from TNFα-induced increase in Angpt-2 transcription and vascular barrier breakdown. Mechanistically, we could exclude canonical Tie2 signalling being responsible but found that three structurally distinct T-type - but not L-type - CC blockers can suppress Angpt-2. Most importantly, experimental increase in intracellular calcium abolished Flunarizine’s effect. Flunarizine was also able to block the injurious increase of Angpt-2 in murine endotoxemia in vivo. This resulted in reduced pulmonary adhesion molecule expression (intercellular adhesion molecule-1) and tissue infiltration of inflammatory cells (Gr-1). Our finding could have therapeutic implications as side effects of Flunarizine are low and specific sepsis therapeutics that target the dysregulated host response are highly desirable

Angiopoietin-2 and biliary diseases: elevated serum, but not bile levels are associated with cholangiocarcinoma.

BACKGROUND:The diagnosis of cholangiocarcinoma (CC) is challenging especially in patients with primary sclerosing cholangitis (PSC) and often delayed due to the lack of reliable markers. Angiopoietin-2 (Angpt-2) has been employed as a biomarker of angiogenesis and might be involved in tumor neoangiogenesis. AIM:To evaluate the diagnostic potential of Angpt-2 as a biomarker to detect patients with CC. METHODS:Bile and serum Angpt-2 levels were measured in patients with CC (n=45), PSC (n=74), CC complicating PSC (CC/PSC) (n=11) and patients with bile duct stones (n=37) in a cross sectional study. Diagnostic accuracy of Angpt-2 was compared to carbohydrate antigen 19-9 (CA19-9). Fluorescent immunohistochemistry from human CC liver tissue samples was performed to localize the origin of Angpt-2. RESULTS:Serum Angpt-2 concentration was significantly elevated in patients with CC compared to control patients (p<0.05). Diagnostic accuracy of Angpt-2 as determined by receiver operating characteristic (ROC) analysis resulted in a higher area under the curve (AUC) value compared to CA19-9 (AUC: 0.85 versus 0.77; 95% confidence interval (CI): 0.74-0.93 versus 0.65-0.87, respectively). Angpt-2 was also detectable in bile, but was not associated with the presence of CC. Immunohistochemistry revealed a strong induction of Angpt-2 expression in the tumor vasculature. CONCLUSIONS:Circulating Angpt-2 in serum might be a promising protein candidate locally derived from the tumor vasculature in patients with CC. Measurement of Angpt-2 in serum may be useful for diagnosis and further clinical management of patients with CC