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The Role of Prominin-1 in the Architecture and Dynamics of Microvilli and Primary CiliaThamm, Kristina 15 January 2018 (has links)
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.
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The Role of Prominin-1 in the Architecture and Dynamics of Microvilli and Primary CiliaThamm, Kristina 15 January 2018 (has links)
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.
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Cell transplantation and gene therapy approaches for the treatment of retinal degenerative disordersEberle, Dominic 09 January 2013 (has links) (PDF)
Photoreceptors are of prime importance for humans, since vision is one of the most important senses for us. In our daily life, where nearly every action is dependent on visual input, an impairment or a loss of eyesight leads to severe disability. With a non-syndromic prevalence of 1:4000, retinitis pigmentosa, a collective term for a group of inherited retinal eye diseases, represents, together with age-related macula degeneration, one of the main causes for visual impairment and blindness in industrialized countries. The dominant reason for vision loss is, in both cases, the irreversible loss of photoreceptor cells located in the outer nuclear layer of the retina. To date, no effective treatment is available to preserve or regain visual function in affected patients. Recent promising strategies for new retinal therapeutical approaches focus on one hand on the development of gene therapies, where an introduced wild-type allele compensates a mutated gene, and on the other hand on cell therapies, where stem or photoreceptor precursor cells (PPCs) are transplanted to the sub-retinal space to replace degenerated host photoreceptors.
The current study is subdivided into three parts, addressing the issue of non-reversible photoreceptor cell loss due to retinal degenerative diseases by investigating in the first two parts new qualitative as well as quantitative approaches in the field of retinal cell therapy, while in the third part an ocular gene therapeutical approach targeting prominin-1, a gene involved in retinal degenerative disorders, was investigated. Briefly, this study shows in the first part, a significant enhancement of the integration rate of PPCs in wild-type host retinas, achieved by pre-transplantational sorting, using the recently discovered PPC - specific cell surface marker CD73. This sets another step further towards retinal cell therapy by increasing the effectiveness of such treatment. Next to this quantitative approach, it is also shown that the quality of transplanted photoreceptor precursor cells is comparable to native photoreceptors by demonstrating, that an indispensable prerequisite of every photoreceptor cell, the outer segment, is developed by transplanted PPCs after proper integration. Importantly, transplanted PPCs develop native outer segments even when not integrated in the host tissue but located in the sub-retinal space, as it is predominantly observed after transplantation into severely degenerated retinas. These results substantiate the feasibility of cell therapeutical treatment of severely degenerated retinas. At the end of this part, it is demonstrated, that outer segments are not formed properly by PPCs transplanted to the vitreal side of the retina. This suggests an influence of signaling molecules, presumably secreted by retinal pigment epithelial cells into the sub-retinal space, on transplanted PPC final differentiation. Since intensive research is done to differentiate stem cells into PPCs for cell therapeutical transplantation, these results may contribute significantly to this research by demonstrating, that factors secreted by the retinal pigment epithelium might play a crucial role for successful stem cell to PPC differentiation.
The last part of my work investigates a gene therapeutical approach to cure inherited retinal degenerative diseases. One gene, where reported mutations cause retinal degeneration in humans is prominin-1, a protein expressed at cell membrane evaginations in a variety of cell types. Interestingly, the prominin-1 knock-out mouse is characterized exclusively by disorganized photoreceptor outer segment formation and progressive retinal degeneration. Successful delivery of a wild-type form of mouse prominin-1 using adeno-associated viral vector transfer, into the photoreceptors of prominin-1 - deficient mice is demonstrated. The divergent results show on one hand a rescue of the thickness of the photoreceptor outer nuclear layer on a short time period (3 weeks post treatment), and on the other hand long-term data (8-10 weeks post treatment) suggests histologically as well as functionally a negative effect on treated photoreceptors. This might be due to effects caused by an over-expression of prominin-1 and will be investigated in future studies. In conclusion, distinct and important investigations were made which contribute significant puzzle pieces to new cell- as well as gene therapeutical approaches for the treatment of retinal degenerative disorders.
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Cell transplantation and gene therapy approaches for the treatment of retinal degenerative disordersEberle, Dominic 21 December 2012 (has links)
Photoreceptors are of prime importance for humans, since vision is one of the most important senses for us. In our daily life, where nearly every action is dependent on visual input, an impairment or a loss of eyesight leads to severe disability. With a non-syndromic prevalence of 1:4000, retinitis pigmentosa, a collective term for a group of inherited retinal eye diseases, represents, together with age-related macula degeneration, one of the main causes for visual impairment and blindness in industrialized countries. The dominant reason for vision loss is, in both cases, the irreversible loss of photoreceptor cells located in the outer nuclear layer of the retina. To date, no effective treatment is available to preserve or regain visual function in affected patients. Recent promising strategies for new retinal therapeutical approaches focus on one hand on the development of gene therapies, where an introduced wild-type allele compensates a mutated gene, and on the other hand on cell therapies, where stem or photoreceptor precursor cells (PPCs) are transplanted to the sub-retinal space to replace degenerated host photoreceptors.
The current study is subdivided into three parts, addressing the issue of non-reversible photoreceptor cell loss due to retinal degenerative diseases by investigating in the first two parts new qualitative as well as quantitative approaches in the field of retinal cell therapy, while in the third part an ocular gene therapeutical approach targeting prominin-1, a gene involved in retinal degenerative disorders, was investigated. Briefly, this study shows in the first part, a significant enhancement of the integration rate of PPCs in wild-type host retinas, achieved by pre-transplantational sorting, using the recently discovered PPC - specific cell surface marker CD73. This sets another step further towards retinal cell therapy by increasing the effectiveness of such treatment. Next to this quantitative approach, it is also shown that the quality of transplanted photoreceptor precursor cells is comparable to native photoreceptors by demonstrating, that an indispensable prerequisite of every photoreceptor cell, the outer segment, is developed by transplanted PPCs after proper integration. Importantly, transplanted PPCs develop native outer segments even when not integrated in the host tissue but located in the sub-retinal space, as it is predominantly observed after transplantation into severely degenerated retinas. These results substantiate the feasibility of cell therapeutical treatment of severely degenerated retinas. At the end of this part, it is demonstrated, that outer segments are not formed properly by PPCs transplanted to the vitreal side of the retina. This suggests an influence of signaling molecules, presumably secreted by retinal pigment epithelial cells into the sub-retinal space, on transplanted PPC final differentiation. Since intensive research is done to differentiate stem cells into PPCs for cell therapeutical transplantation, these results may contribute significantly to this research by demonstrating, that factors secreted by the retinal pigment epithelium might play a crucial role for successful stem cell to PPC differentiation.
The last part of my work investigates a gene therapeutical approach to cure inherited retinal degenerative diseases. One gene, where reported mutations cause retinal degeneration in humans is prominin-1, a protein expressed at cell membrane evaginations in a variety of cell types. Interestingly, the prominin-1 knock-out mouse is characterized exclusively by disorganized photoreceptor outer segment formation and progressive retinal degeneration. Successful delivery of a wild-type form of mouse prominin-1 using adeno-associated viral vector transfer, into the photoreceptors of prominin-1 - deficient mice is demonstrated. The divergent results show on one hand a rescue of the thickness of the photoreceptor outer nuclear layer on a short time period (3 weeks post treatment), and on the other hand long-term data (8-10 weeks post treatment) suggests histologically as well as functionally a negative effect on treated photoreceptors. This might be due to effects caused by an over-expression of prominin-1 and will be investigated in future studies. In conclusion, distinct and important investigations were made which contribute significant puzzle pieces to new cell- as well as gene therapeutical approaches for the treatment of retinal degenerative disorders.
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