Number and Brightness Analysis of LRRK2 Oligomerization in Live Cells

James, Nicholas G.; Digman, Michelle A.; Gratton, Enrico; Baryłko, Barbara; Ding, Xiaodong; Albanesi, Joseph P.; Goldberg, Matthew S.; Jameson, David M.

doi:10.1016/j.bpj.2012.04.046

Cited by 70 publications

(72 citation statements)

References 17 publications

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“…N&B analysis was carried out in order to obtain information about the apparent oligomerization state of mRFP-Lact-C2 (49)(50)(51)(52). Since mRFP-Lact-C2 binds specifically to PS, the presence of PS-rich platforms (i.e., capable of binding more than one mRFPLact-C2 molecule) should be evident as an increased apparent multimerization of the protein (i.e., more mRFP-Lact-C2 molecules diffusing together as a single unit).…”

Section: Resultsmentioning

confidence: 99%

Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes

Bobone

Hilsch

Storm

et al. 2017

J Virol

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Influenza A virus matrix protein 1 (M1) is an essential component involved in the structural stability of the virus and in the budding of new virions from infected cells. A deeper understanding of the molecular basis of virion formation and the budding process is required in order to devise new therapeutic approaches. We performed a detailed investigation of the interaction between M1 and phosphatidylserine (PS) (i.e., its main binding target at the plasma membrane [PM]), as well as the distribution of PS itself, both in model membranes and in living cells. To this end, we used a combination of techniques, including Förster resonance energy transfer (FRET), confocal microscopy imaging, raster image correlation spectroscopy, and number and brightness (N&B) analysis. Our results show that PS can cluster in segregated regions in the plane of the lipid bilayer, both in model bilayers constituted of PS and phosphatidylcholine and in living cells. The viral protein M1 interacts specifically with PS-enriched domains, and such interaction in turn affects its oligomerization process. Furthermore, M1 can stabilize PS domains, as observed in model membranes. For living cells, the presence of PS clusters is suggested by N&B experiments monitoring the clustering of the PS sensor lactadherin. Also, colocalization between M1 and a fluorescent PS probe suggest that, in infected cells, the matrix protein can specifically bind to the regions of PM in which PS is clustered. Taken together, our observations provide novel evidence regarding the role of PS-rich domains in tuning M1-lipid and M1-M1 interactions at the PM of infected cells.IMPORTANCE Influenza virus particles assemble at the plasma membranes (PM) of infected cells. This process is orchestrated by the matrix protein M1, which interacts with membrane lipids while binding to the other proteins and genetic material of the virus. Despite its importance, the initial step in virus assembly (i.e., M1-lipid interaction) is still not well understood. In this work, we show that phosphatidylserine can form lipid domains in physical models of the inner leaflet of the PM. Furthermore, the spatial organization of PS in the plane of the bilayer modulates M1-M1 interactions. Finally, we show that PS domains appear to be present in the PM of living cells and that M1 seems to display a high affinity for them.KEYWORDS influenza, assembly, confocal microscopy, fluorescence image analysis, lipid rafts, matrix protein, model membranes, phosphatidylserine, plasma membrane I nfluenza A virus (IAV) is a major burden to human health and remains a prominent cause of mortality worldwide (1, 2). Despite significant research efforts, detailed knowledge about the molecular mechanisms involved in IAV infection is still limited. A deeper understanding of the virus machinery and its interaction with the host is

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Section: Resultsmentioning

confidence: 99%

Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes

Bobone

Hilsch

Storm

et al. 2017

J Virol

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“…G2019S carriers exhibit a phenotype nearly indistinguishable from idiopathic PD, with a late onset and often with LBs pathology [18]. Accumulating evidence indicates that LRRK2 is associated with membrane compartments [19,20] where it phosphorylates key proteins involved in membrane remodeling [17,21,22] and regulates different processes including autophagylysosome pathway [23], vesicular trafficking and protein sorting [14,24]. However, Lrrk2 -/-mice or rats show a normal dopaminergic system, with subtle or no alterations in the number of dopaminergic neurons and in the levels of striatal dopamine [25].…”

Section: Introductionmentioning

confidence: 99%

LRRK2 deficiency impacts ceramide metabolism in brain

Ferrazza

Cogo

Melrose

et al. 2016

Biochemical and Biophysical Research Communications

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Mutations in LRRK2 gene cause inherited Parkinson's disease (PD) and variations around LRRK2 act as risk factor for disease. Similar to sporadic disease, LRRK2-linked cases show late onset and, typically, the presence of proteinaceous inclusions named Lewy bodies (LBs) in neurons. Recently, defects on ceramide (Cer) metabolism have been recognized in PD. In particular, heterozygous mutations in the gene encoding for glucocerebrosidase (GBA1), a lysosomal enzyme converting glucosyl-ceramides (Glc-Cer) into Cer, increase the risk of developing PD. Although several studies have linked LRRK2 with membrane-related processes and autophagic-lysosomal pathway regulation, whether this protein impinges on the Cer pathway has not been addressed. Here, using a targeted lipidomics approach, we report an altered sphingolipid composition in Lrrk2 -/-mouse brains. In particular, we observe a significant increase of Cer levels in Lrrk2 -/-mice and direct effects on GBA1. Collectively, our results suggest a link between LRRK2 and Cer metabolism, providing new insights into the possible role of this protein in sphingolipids metabolism, with implications for PD therapeutics.

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“…Diverse processes such as chemical kinetics, molecular diffusion, protein transport, protein oligomerization, molecular interactions, and stoichiometries can now be followed with single molecule sensitivity and at microsecond timescales in biological systems. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] These microscopy-based techniques that make up this burgeoning field at the interface of biology and physics are collectively called fluorescence fluctuation spectroscopy (FFS) techniques (for review, see Refs. [19][20][21].…”

Section: Introductionmentioning

confidence: 99%

Measuring protein dynamics in live cells: protocols and practical considerations for fluorescence fluctuation microscopy

Youker

Teng

2014

J. Biomed. Opt

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Quantitative analysis of protein complex stoichiometries and mobilities are critical for elucidating the mechanisms that regulate cellular pathways. Fluorescence fluctuation spectroscopy (FFS) techniques can measure protein dynamics, such as diffusion coefficients and formation of complexes, with extraordinary precision and sensitivity. Complete calibration and characterization of the microscope instrument is necessary in order to avoid artifacts during data acquisition and to capitalize on the full capabilities of FFS techniques. We provide an overview of the theory behind FFS techniques, discuss calibration procedures, provide protocols, and give practical considerations for performing FFS experiments. One important parameter recovered from FFS measurements is the relative molecular brightness that can correlate with oligomerization. Three methods for measuring molecular brightness (fluorescence correlation spectroscopy, photon-counting histogram, and number and brightness analysis) recover similar values when measuring samples under ideal conditions in vitro. However, examples are given illustrating that these different methods used for calculating molecular brightness of fluorescent molecules in cells are not always equivalent. Methods relying on spot measurements are more prone to bleaching and movement artifacts that can lead to underestimation of brightness values. We advocate for the use of multiple FFS techniques to study molecular brightnesses to overcome and compliment limitations of individual techniques.

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Number and Brightness Analysis of LRRK2 Oligomerization in Live Cells

Cited by 70 publications

References 17 publications

Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes

Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes

LRRK2 deficiency impacts ceramide metabolism in brain

Measuring protein dynamics in live cells: protocols and practical considerations for fluorescence fluctuation microscopy

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