scholarly journals Fluctuation Imaging of LRRK2 Reveals that the G2019S Mutation Alters Spatial and Membrane Dynamics

Molecules ◽  
2020 ◽  
Vol 25 (11) ◽  
pp. 2561
Author(s):  
Bethany J. Sanstrum ◽  
Brandee M. S. S. Goo ◽  
Diana Z. Y. Holden ◽  
Donovan D. Delgado ◽  
Thien P. N. Nguyen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Dynamics ◽  
Vesicular Trafficking ◽  
Leucine Rich Repeat ◽  
Membrane Recruitment ◽  
G2019s Mutation ◽  
Lrrk2 Gene ◽  
Endocytic Protein ◽  
Sporadic Parkinson’S Disease ◽  
Protein Recruitment

Mutations within the Leucine-Rich Repeat Kinase 2 (LRRK2) gene are the most common genetic cause of autosomal and sporadic Parkinson’s disease (PD). LRRK2 is a large multidomain kinase that has reported interactions with several membrane proteins, including Rab and Endophilin, and has recently been proposed to function as a regulator of vesicular trafficking. It is unclear whether or how the spatiotemporal organization of the protein is altered due to LRRK2 activity. Therefore, we utilized fluctuation-based microscopy along with FLIM/FRET to examine the cellular properties and membrane recruitment of WT LRRK2-GFP (WT) and the PD mutant G2019S LRRK2-GFP (G2019S). We show that both variants can be separated into two distinct populations within the cytosol; a freely diffusing population associated with monomer/dimer species and a slower, likely vesicle-bound population. G2019S shows a significantly higher propensity to self-associate in both the cytosol and membrane regions when compared to WT. G2019S expression also resulted in increased hetero-interactions with Endophilin A1 (EndoA1), reduced cellular vesicles, and altered clathrin puncta dynamics associated with the plasma membrane. This finding was associated with a reduction in transferrin endocytosis in cells expressing G2019S, which indicates disruption of endocytic protein recruitment near the plasma membrane. Overall, this study uncovered multiple dynamic alterations to the LRRK2 protein as a result of the G2019S mutation—all of which could lead to neurodegeneration associated with PD.

scholarly journals High temporal resolution reveals simultaneous plasma membrane recruitment of the TPLATE complex subunits

2020 ◽  
Author(s):  
Jie Wang ◽  
Evelien Mylle ◽  
Alexander Johnson ◽  
Nienke Besbrugge ◽  
Geert De Jaeger ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Temporal Resolution ◽  
Live Cell Imaging ◽  
Adaptor Protein ◽  
High Temporal Resolution ◽  
Spatiotemporal Resolution ◽  
Membrane Recruitment ◽  
Localization Analysis ◽  
Dynamic Assembly ◽  
Protein Recruitment

AbstractThe TPLATE complex (TPC) is a key endocytic adaptor protein complex in plants. TPC contains six evolutionary conserved subunits and two plant specific subunits, AtEH1/Pan1 and AtEH2/Pan1, which are not associated with the hexameric subcomplex in the cytoplasm. To investigate the dynamic assembly of the octameric TPC at the plasma membrane (PM), we performed state-of-the-art dual-color live cell imaging at physiological and a lowered temperature. Our data show that lowering the temperature slows down endocytosis and thereby enhances the temporal resolution of the differential recruitment of endocytic components. Under both normal and lowered temperature conditions, the core TPC subunit TPLATE, and the AtEH/Pan1 proteins, exhibited simultaneous recruitment at the PM. These results, together with our co-localization analysis of different TPC subunits, allow us to conclude that in plant cells, TPC is not recruited to the PM sequentially but as an octameric complex.One sentence summaryLowering the temperature increases spatiotemporal resolution of protein recruitment at the plasma membrane.

Cellular functions of LRRK2 implicate vesicular trafficking pathways in Parkinson's disease

2016 ◽  
Vol 44 (6) ◽  
pp. 1603-1610 ◽  
Author(s):  
Mark R. Cookson
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Intracellular Trafficking ◽  
Small Gtpases ◽  
Vesicular Trafficking ◽  
Leucine Rich Repeat ◽  
Cellular Functions ◽  
Lrrk2 Gene ◽  
Rab Family ◽  
Cellular Compartments

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene, associated with Parkinson's disease, have been shown to affect intracellular trafficking pathways in a variety of cells and organisms. An emerging theme is that LRRK2 can bind to multiple membranous structures in cells, and several recent studies have suggested that the Rab family of small GTPases might be important in controlling the recruitment of LRRK2 to specific cellular compartments. Once localized to membranes, LRRK2 then influences downstream events, evidenced by changes in the autophagy–lysosome pathway. Here, I will discuss available evidence that supports or challenges this outline, with a specific emphasis on those aspects of LRRK2 function that have been controversial or remain to be fully clarified.

scholarly journals LRRK2 Screening in a Canadian Parkinson's Disease Cohort

2007 ◽  
Vol 34 (3) ◽  
pp. 336-338 ◽  
Author(s):  
D. A. Grimes ◽  
L. Racacho ◽  
F. Han ◽  
M. Panisset ◽  
D. E. Bulman
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
High Performance Liquid Chromatography ◽  
High Performance ◽  
Sequence Variants ◽  
Leucine Rich Repeat ◽  
Novel Mutations ◽  
Intronic Sequence ◽  
G2019s Mutation ◽  
Lrrk2 Gene

Background:Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) have become the most common known cause for developing Parkinson's disease. The frequency of mutations described in the literature varies widely depending on the population studied with most reports focusing only on screening for the most common G2019S mutation in exon 41.Methods:In this study seven exons (19, 24, 25, 31, 35, 38, and 41) in LRRK2 where mutations have been reported were screened in 230 unselected Parkinson's disease patients using denaturing high-performance liquid chromatography.Results:The sequencing of samples with heteroduplex profiles revealed five novel and two known intronic sequence variants. In our cohort, we were unable to detect any of the known mutations in these exons or identify novel mutations within the LRRK2 gene.Conclusions:Therefore, despite the availability of diagnostic LRRK2 genetic testing it is unlikely to yield a positive result in this population.

scholarly journals Cargo-mediated recruitment of the endocytic adaptor protein Sla1 in S. cerevisiae

2020 ◽  
Vol 133 (19) ◽  
pp. jcs247684
Author(s):  
Thomas O. Tolsma ◽  
Hallie P. Febvre ◽  
Deanna M. Olson ◽  
Santiago M. Di Pietro
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Point Mutations ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Coat Proteins ◽  
Plasma Membrane Proteins ◽  
Membrane Recruitment ◽  
Surface Localization ◽  
Protein Recruitment

ABSTRACTEndocytosis of plasma membrane proteins is mediated by their interaction with adaptor proteins. Conversely, emerging evidence suggests that adaptor protein recruitment to the plasma membrane may depend on binding to endocytic cargo. To test this idea, we analyzed the yeast adaptor protein Sla1, which binds membrane proteins harboring the endocytic signal NPFxD via the Sla1 SHD1 domain. Consistently, SHD1 domain point mutations that disrupted NPFxD binding caused a proportional reduction in Sla1–GFP recruitment to endocytic sites. Furthermore, simultaneous SHD1 domain point mutation and deletion of the C-terminal LxxQxTG repeat (SR) region linking Sla1 to coat proteins Pan1 and End3 resulted in total loss of Sla1–GFP recruitment to the plasma membrane. These data suggest that multiple interactions are needed for recruitment of Sla1 to the membrane. Interestingly, a Sla1 fragment containing just the third SH3 domain, which binds ubiquitin, and the SHD1 domain displayed broad surface localization, suggesting plasma membrane recruitment is mediated by interaction with both NPFxD-containing and ubiquitylated plasma membrane proteins. Our results also imply that a Sla1 NPF motif adjacent to the SR region might regulate the Sla1–cargo interaction, mechanistically linking Sla1 cargo binding to endocytic site recruitment.

scholarly journals Measurement of dynamic membrane mechanosensation using optical tweezers

2021 ◽  
Author(s):  
Xuanling Li ◽  
Xing Liu ◽  
Xiaoyu Song ◽  
Yinmei Li ◽  
Ming Li ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Optical Tweezers ◽  
Environmental Changes ◽  
Quantitative Measure ◽  
Relaxation Curve ◽  
Membrane Dynamics ◽  
Molecular Organization ◽  
Cytoskeletal Remodeling ◽  
Relaxation Curves ◽  
Membrane Tether

Abstract Many cellular processes are orchestrated by dynamic changes in the plasma membrane to form membrane projections and endocytic vesicles in response to extracellular environmental changes. Our previous studies show that ARF6-ACAP4-ezrin signaling regulates membrane dynamics and curvature in response to EGF stimulation. However, there is no quantitative measurement to relate molecular organization of membrane cytoskeletal remodeling to stimulus-elicited mechanosensation on the plasma membrane. Optical tweezers is a powerful tool in the study of membrane tension. Comparing to pulling out an entire membrane tether at one time, the step-like method is more efficient because multiple relaxation curves can be obtained from one membrane tether. Fewer models describe relaxation curves to characterize mechanical properties of cell membrane. Here we establish a new method to measure the membrane relaxation curve of HeLa cells judged by the relationship between membrane tether diameter and tensions. We obtained effective viscosities and static tensions by fitting relaxation curves to our model. We noticed the delicate structure of relaxation curves contains information of cytoskeletal remodeling and lateral protein diffusion. Our study established a quantitative measure to characterize the mechanosensation of epithelial cells in response to stimulus-elicited membrane dynamics.

scholarly journals Endocytosis: a review of mechanisms and plasma membrane dynamics

1983 ◽  
Vol 210 (1) ◽  
pp. 1-13 ◽  
Author(s):  
J M Besterman ◽  
R B Low
Keyword(s):  
Plasma Membrane ◽  
Membrane Dynamics

scholarly journals HIV-1 requires Arf6-mediated membrane dynamics to efficiently enter and infect T lymphocytes

2011 ◽  
Vol 22 (8) ◽  
pp. 1148-1166 ◽  
Author(s):  
Laura García-Expósito ◽  
Jonathan Barroso-González ◽  
Isabel Puigdomènech ◽  
José-David Machado ◽  
Julià Blanco ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
T Lymphocytes ◽  
Viral Entry ◽  
Membrane Trafficking ◽  
Membrane Dynamics ◽  
Viral Fusion ◽  
Viral Attachment ◽  
Immunodeficiency Virus ◽  
Vesicular Stomatitis ◽  
Hiv 1

As the initial barrier to viral entry, the plasma membrane along with the membrane trafficking machinery and cytoskeleton are of fundamental importance in the viral cycle. However, little is known about the contribution of plasma membrane dynamics during early human immunodeficiency virus type 1 (HIV-1) infection. Considering that ADP ribosylation factor 6 (Arf6) regulates cellular invasion via several microorganisms by coordinating membrane trafficking, our aim was to study the function of Arf6-mediated membrane dynamics on HIV-1 entry and infection of T lymphocytes. We observed that an alteration of the Arf6–guanosine 5′-diphosphate/guanosine 5′-triphosphate (GTP/GDP) cycle, by GDP-bound or GTP-bound inactive mutants or by specific Arf6 silencing, inhibited HIV-1 envelope–induced membrane fusion, entry, and infection of T lymphocytes and permissive cells, regardless of viral tropism. Furthermore, cell-to-cell HIV-1 transmission of primary human CD4+T lymphocytes was inhibited by Arf6 knockdown. Total internal reflection fluorescence microscopy showed that Arf6 mutants provoked the accumulation of phosphatidylinositol-(4,5)-biphosphate–associated structures on the plasma membrane of permissive cells, without affecting CD4-viral attachment but impeding CD4-dependent HIV-1 entry. Arf6 silencing or its mutants did not affect fusion, entry, and infection of vesicular stomatitis virus G–pseudotyped viruses or ligand-induced CXCR4 or CCR5 endocytosis, both clathrin-dependent processes. Therefore we propose that efficient early HIV-1 infection of CD4+T lymphocytes requires Arf6-coordinated plasma membrane dynamics that promote viral fusion and entry.

Insulin modulates PC-1 processing and recruitment in cultured human cells

2003 ◽  
Vol 284 (3) ◽  
pp. E514-E520 ◽  
Author(s):  
C. Menzaghi ◽  
R. Di Paola ◽  
G. Baj ◽  
A. Funaro ◽  
A. Arnulfo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Plasma Membrane ◽  
Intracellular Trafficking ◽  
Posttranslational Processing ◽  
Phosphatidylinositol 3 Kinase ◽  
S6 Kinase ◽  
Fourfold Increase ◽  
Membrane Recruitment ◽  
Insulin Receptor Signaling ◽  
Cultured Human Cells

We evaluated whether insulin signaling modulates plasma cell glycoprotein (PC-1) plasma membrane recruitment, posttranslational processing, and gene expression in human cultured cell lines. Insulin induced a fourfold increase ( P < 0.01) of membrane PC-1 expression by rapid and sensitive mechanism(s). This effect was reduced ( P < 0.05–0.01) by inhibition of phosphatidylinositol 3-kinase (200 nmol/l wortmannin) and S6 kinase (50 nmol/l rapamycin) activities and intracellular trafficking (50 μmol/l monensin) and was not accompanied by PC-1 gene expression changes. Moreover, at Western blot, insulin elicited the appearance, in both plasma membrane and cytosol, of a PC-1-related 146-kDa band (in addition to bands of 163, 117, 106, and 97 kDa observed also in absence of insulin) that was sensitive to endoglycosidase H. Finally, inhibition of PC-1 translocation to plasma membrane, by wortmannin pretreatment, increases insulin-stimulated receptor autophosphorylation. Our data indicate that insulin stimulates PC-1 posttranslational processing and translocation to the plasma membrane, which in turn impairs insulin receptor signaling. Bidirectional cross talk between insulin and PC-1, therefore, takes place, which may be part of the hormone self-desensitization mechanism.

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