Number and brightness analysis of LRRK2 oligomerization in live cells

Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein that contains enzymatically functional GTPase and kinase domains. Several noncoding LRRK2 gene polymorphisms have been associated with susceptibility to Parkinson's disease (PD), Crohn's disease, and leprosy. Many LRRK2 coding polymorphisms have been associated with or causally linked to PD. The G2019S point mutation within the LRRK2 kinase domain is the most common cause of familial PD. The G2019S mutation appears to alter LRRK2 kinase activity. Some but not all studies have reported that LRRK2 kinase activity is dependent upon LRRK2 dimerization and membrane localization. It is important to define the oligomeric state(s) of LRRK2 in living cells, which to date have only been characterized in vitro. Here we use confocal and total internal reflection microscopy coupled with number and brightness analysis to study the oligomeric states of LRRK2 within the cytosol and on the plasma membrane of live CHO-K1 cells. Our results show, for the first time to our knowledge, that LRRK2 is predominantly monomeric throughout the cytosol of living cells, but attains predominately higher oligomeric states in the plasma membrane.     

The mitochondrial permeability transition pore (PTP) - an example of multiple molecular exaptation?

The mitochondrial permeability transition (PT) is a well-recognized phenomenon that allows mitochondria to undergo a sudden increase of permeability to solutes with molecular mass ≤ 1500Da, leading to organelle swelling and structural modifications. The relevance of PT relies on its master role in the manifestation of programmed cell death (PCD). This function is performed by a mega-channel (in some cases inhibited by cyclosporin A) named permeability transition pore (PTP), whose function could derive from the assembly of different mitochondrial proteins. In this paper we examine the distribution and characteristics of PTP in mitochondria of eukaryotic organisms so far investigated in order to draw a hypothesis on the mechanism of its evolution. As a result, we suggest that PTP may have arisen as a new function linked to a multiple molecular exaptation of different mitochondrial proteins, even though they could nevertheless still play their original role. Furthermore, we suggest that the early appearance of PTP could have had a crucial role in the establishment of endosymbiosis in eukaryotic cells, by the coordinated balancing of ATP production by glycolysis (performed by the primary phagocyte) and oxidative phosphorylation (accomplished by the endosymbiont). Indeed, we argue on the possibility that this new energetic equilibrium could have opened the way to the subsequent evolution toward metazoans.     

2D-DIGE analysis of ovarian cancer cell responses to cytotoxic gold compounds

Cytotoxic gold compounds hold today great promise as new pharmacological agents for treatment of human ovarian carcinoma; yet, their mode of action is still largely unknown. To shed light on the underlying molecular mechanisms, we performed 2D-DIGE analysis to identify differential protein expression in a cisplatin-sensitive human ovarian cancer cell line (A2780/S) following treatment with two representative gold(iii) complexes that are known to be potent antiproliferative agents, namely AuL12 and Au(2)Phen. Software analysis using DeCyder was performed and few differentially expressed protein spots were visualized between the three examined settings after 24 h exposure to the cytotoxic compounds, implying that cellular damage at least during the early phases of exposure is quite limited and selective, reflecting the attempts of the cell to repair damage and to survive the insult. The potential of novel proteomic methods to disclose mechanistic details of cytotoxic metallodrugs is herein further highlighted. Different patterns of proteomic changes were highlighted for the two metallodrugs with only a few perturbed protein spots in common. Using MALDI-TOF MS and ESI-Ion trap MS/MS, several differentially expressed proteins were identified. Two of these were validated by western blotting: Ubiquilin-1, responsible for inhibiting degradation of proteins such as p53 and NAP1L1, a candidate marker identified in primary tumors. Ubiquilin-1 resulted over-expressed following both treatments and NAP1L1 was down-expressed in AuL12-treated cells in comparison with control and with Au(2)Phen-treated cells. In conclusion, we performed a comprehensive analysis of proteins regulated by AuL12 and Au(2)Phen, providing a useful insight into their mechanisms of action.     

Visualization and quantification of cardiac mitochondrial protein clusters with STED microscopy

The visualization and quantification of mitochondria-associated proteins with high power microscopy methods is of particular interest to investigate protein architecture in this organelle. We report the usage of a custom-made STimulated Emission Depletion (STED) fluorescence nanoscope with ~30nm lateral resolution for protein mapping of Percoll-purified viable mitochondria from murine heart. Using this approach, we were able to quantify and resolve distinct protein clusters within mitochondria; specifically, cytochrome c oxidase subunit 2 is distributed in clusters of ~28nm; whereas the voltage dependent anion channel 1 displays three size distributions of ~33, ~55 and ~83nm.     

Cross-linking of phospholipid membranes is a conserved property of calcium-sensitive synaptotagmins

Synaptotagmins are vesicular proteins implicated in many membrane trafficking events. They are highly conserved in evolution and the mammalian family contains 16 isoforms. We now show that the tandem C2 domains of several calcium-sensitive synaptotagmin isoforms tested, including Drosophila synaptotagmin, rapidly cross-link phospholipid membranes. In contrast to the tandem structure, individual C2 domains failed to trigger membrane cross-linking in several novel assays. Large-scale liposomal aggregation driven by tandem C2 domains in response to calcium was confirmed by the following techniques: turbidity assay, dynamic light-scattering and both confocal and negative stain electron microscopy. Firm cross-linking of membranes was evident from laser trap experiments. High-resolution cryo-electron microscopy revealed that membrane cross-linking by tandem C2 domains results in a constant distance of ∼9 nm between the apposed membranes. Our findings show the conserved nature of this important property of synaptotagmin, demonstrate the significance of the tandem C2 domain structure and provide a plausible explanation for the accelerating effect of synaptotagmins on membrane fusion.     

Protein osmotic pressure modulates actin filament length distribution

1.

The changes of the macromolecular osmotic pressure associated with F-actin solutions are related to the changes of the free energy of the free actin monomers.

2.

By making use of the model of Biron et al. [2006. Inter-filament attractions narrow the length distribution of actin filaments. Europhys. Lett. 73, 464-470], the changes of the free energy of the free actin monomers are related to the changes of the length distribution of the actin filaments.

On these bases, we propose that the length distribution of the actin filaments is regulated by (a) the free energy of hydrolysis of ATP and (b) the macromolecular osmotic pressure.

a.

While the free energy of hydrolysis of ATP tends to zero, the length distribution of the actin filaments shifts from an exponential curve to a straight line parallel to the abscissa axis (i.e. the concentration of the actin filaments becomes independent of their length). In the mean time, the total energy of the actin filaments reaches a minimum.

b.

With the increase of the macromolecular osmotic pressure the free energy of the actin monomers increases and a break is introduced in the curve that describes the length distribution of the actin filaments; the break is located at the mean length of the filaments.

     

High content analysis of gamma-secretase activity reveals variable dominance of presenilin mutations linked to familial Alzheimer's disease

gamma-Secretase mediates the intramembranous proteolysis of amyloid precursor protein (APP), Notch and other cellular substrates and is considered a prime pharmacological target in the development of therapeutics for Alzheimer's disease (AD). We describe here an efficient, new, simple, sensitive and rapid assay to quantify gamma-secretase activity in living cells by flow cytometry using two membrane-bound fluorescent probes, APP-GFP or C99-GFP, as substrates for gamma-secretase. The principle of the assay is based on the fact that the soluble intracellular domain of GFP-tagged APP (AICD-GFP) is released from the membrane into the cytosol following gamma-secretase cleavage. Using this feature, enzymatic activity of gamma-secretase could be deduced from the extent of the membrane retention of the probe observed after plasma membrane permeabilization and washout of the cleaved fraction. By applying two well-known gamma-secretase inhibitors (DAPT and L-685,458), we validated our assay showing that the positional GFP-based probes for gamma-secretase activity behave properly when expressed in different cell lines, providing the basis for the further development of a high-throughput and high content screening for AD targeted drug discovery. Moreover, by co-expression of different familial AD-linked mutated forms of presenilin--the key component of the gamma-secretase complex--in cells devoid of any endogenous gamma-secretase, our method allowed us to evaluate in situ the contribution of different presenilin variants to the modulation of the enzyme.     

Yeast ribosomal stalk heterogeneity in vivo shown by two-photon FCS and molecular brightness analysis

The stalk of Saccharomyces cerevisiae ribosomes contains, on average, five distinct proteins, namely P0 and four acidic proteins, P1α, P1β, P2α, and P2β. Each ribosome contains only one copy of P0, but the distribution of the acidic proteins among the ribosome population in vivo has not been determined. Using two-photon fluorescence correlation spectroscopy and scanning FCS, on cells expressing EGFP-tagged P0, P1, and P2 proteins, we show, with brightness analysis, that individual yeast ribosomes in vivo are compositionally heterogeneous in regard to P1α, P1β, P2α, and P2β. These results are relevant to the hypothesis, based on in vitro studies, that the overall cellular pattern of expressed proteins can be determined by the distribution of the stalk proteins among the ribosome population.     

The phasor approach to fluorescence lifetime imaging analysis

Changing the data representation from the classical time delay histogram to the phasor representation provides a global view of the fluorescence decay at each pixel of an image. In the phasor representation we can easily recognize the presence of different molecular species in a pixel or the occurrence of fluorescence resonance energy transfer. The analysis of the fluorescence lifetime imaging microscopy (FLIM) data in the phasor space is done observing clustering of pixels values in specific regions of the phasor plot rather than by fitting the fluorescence decay using exponentials. The analysis is instantaneous since is not based on calculations or nonlinear fitting. The phasor approach has the potential to simplify the way data are analyzed in FLIM, paving the way for the analysis of large data sets and, in general, making the FLIM technique accessible to the nonexpert in spectroscopy and data analysis.     

Phosphorylation regulates the assembly of chloroplast import machinery

Chloroplast function depends on the translocation of cytosolically synthesized precursor proteins into the organelle. The recognition and transfer of most precursor proteins across the outer membrane depend on a membrane inserted complex. Two receptor components of this complex, Toc34 and Toc159, are GTPases, which can be phosphorylated by kinases present in the hosting membrane. However, the physiological function of phosphorylation is not yet understood in detail. It is demonstrated that both receptors are phosphorylated within their G-domains. In vitro, the phosphorylation of Toc34 disrupts both homo- and heterodimerization of the G-domains as determined using a phospho-mimicking mutant. In endogenous membranes this mutation or phosphorylation of the wild-type receptor disturbs the association of Toc34, but not of Toc159 with the translocation pore. Therefore, phosphorylation serves as an inhibitor for the association of Toc34 with other components of the complex and phosphorylation can now be discussed as a mechanism to exchange different isoforms of Toc34 within this ensemble.