The C-terminus of the oncoprotein TGAT is necessary for plasma membrane association and efficient RhoA-mediated signaling.

BACKGROUND: Rho guanine exchange factors (RhoGEFs) control cellular processes such as migration, adhesion and proliferation. Alternative splicing of the RhoGEF Trio produces TGAT. The RhoGEF TGAT is an oncoprotein with constitutive RhoGEF activity. We investigated whether the subcellular location of TGAT is critical for its RhoGEF activity. METHODS: Since plasma membrane associated RhoGEFs are particularly effective at activating RhoA, plasma membrane localization of TGAT was examined. To this end, we developed a highly sensitive image analysis method to quantitatively measure plasma membrane association. The method requires a cytoplasmic marker and a plasma membrane marker, which are co-imaged with the tagged protein of interest. Linear unmixing is performed to determine the plasma membrane and cytoplasmic component in the fluorescence signal of protein of interest. RESULTS: The analysis revealed that wild-type TGAT is partially co-localized with the plasma membrane. Strikingly, cysteine TGAT-mutants lacking one or more putative palmitoylation sites in the C-tail, still showed membrane association. In contrast, a truncated variant, lacking the last 15 amino acids, TGATΔ15, lost membrane association. We show that membrane localization of TGAT was responsible for high RhoGEF activity by using a RhoA FRET-sensor and by determining F-actin levels. Mutants of TGAT that still maintained membrane association showed similar activity as wild-type TGAT. In contrast, the activity was abrogated for the cytoplasmic TGATΔ15 variant. Synthetic recruitment of TGATΔ15 to membranes confirmed that TGAT effectively activates RhoA at the plasma membrane. CONCLUSION: Together, these results show that membrane association of TGAT is critical for its activity.

Effect of fixation procedures on the fluorescence lifetimes of Aequorea victoria derived fluorescent proteins.

Fluorescence lifetime imaging microscopy can be used to study protein-protein interactions by Förster Resonance Energy Transfer or to perform lifetime-based multiplexing. Fixation of samples with cells producing fluorescent fusion proteins is commonly used for preservation of samples and for staining with membrane impermeable reagents such as antibodies. However, the effect of fixation methods and mounting media on fluorescence lifetime is poorly documented so far. Here, we demonstrate that fixation by formaldehyde or methanol itself does not affect the lifetime of fluorescent proteins produced in cells but that several widely used mounting media decrease the fluorescence lifetime by up to 20%. It is shown that fixed cells producing Aequorea victoria derived fluorescent proteins mounted in Tris buffer have fluorescence lifetimes indistinguishable from values measured in living cells. Tris buffer also allows accurate Förster Resonance Energy Transfer quantification in fixed cells, as shown with an mTurquoise2-SYFP2 fusion protein. Moreover, identical lifetime contrasts are measured in living and fixed cells mounted in Tris buffer after introducing a single plasmid expressing two lifetime variants of cyan fluorescent proteins, each targeted to different locations in the cell. Our findings will aid the preparation of fixed cells producing fluorescent proteins for reliable measurement of fluorescence lifetimes for Förster Resonance Energy Transfer determination, lifetime based multiplexing and for instrument calibration for standardization purposes.

An introduction to fluorescence imaging techniques geared towards biosensor applications.

After providing a brief overview of the basics of fluorescence and FRET, this chapter discusses the most commonly used methods to record FRET. Emphasis is on microscopy methods that are widely used for biosensor imaging. We cover choice of instruments, describe various ways to detect FRET based on intensity as well as on donor lifetime, and provide some guidelines to match particular recording methods with specific scientific experiments. We end with an extensive discussion on further practical considerations that may greatly affect the success of the experiments.

Understanding why older people develop a wish to die: a qualitative interview study.

BACKGROUND: Quantitative studies in several European countries showed that 10-20% of older people have or have had a wish to die. AIMS: To improve our understanding of why some older people develop a wish to die. METHODS: In-depth interviews with people with a wish to die (n = 31) were carried out. Through open coding and inductive analysis, we developed a conceptual framework to describe the development of death wishes. Respondents were selected from two cohort studies. RESULTS: The wish to die had either been triggered suddenly after traumatic life events or had developed gradually after a life full of adversity, as a consequence of aging or illness, or after recurring depression. The respondents were in a situation they considered unacceptable, yet they felt they had no control to change their situation and thus progressively "gave up" trying. Recurring themes included being widowed, feeling lonely, being a victim, being dependent, and wanting to be useful. Developing thoughts about death as a positive thing or a release from problems seemed to them like a way to reclaim control. CONCLUSIONS: People who wish to die originally develop thoughts about death as a positive solution to life events or to an adverse situation, and eventually reach a balance of the wish to live and to die.

Recruiting end-of-life cancer patients in the Netherlands for a study on suffering and euthanasia requests.

BACKGROUND: In the Netherlands, GPs performed euthanasia or physician-assisted suicide (EAS) in ∼1 of 10 end-of-life cancer patients in their care. Of all explicit requests for EAS directed at GPs, ∼44% resulted in EAS. However, the suffering of patients who do and do not request EAS has never been studied. An important barrier for such research is the low prevalence of end-of-life cancer patients per practice (on average two/year). We studied whether it is possible to recruit end-of-life cancer patients, following-up for requests for EAS (if any), in an interview study in general practice, whether selection occurred and which were the threats and opportunities to recruitment. Our target was to recruit at least 50 patients. METHODS: Characteristics of all eligible patients were monitored. RESULTS: One in every three eligible patients were recruited by 44 GPs in a 3-year inclusion period, resulting in 64 patients in the interview study with follow-up until death. The prevalence of explicit requests for EAS was higher (27%; P = 0.026) in the interview sample, and the presence of a depressed mood according to the GP was lower (5%; P = 0.013) than in the sample with eligible but not participating patients. CONCLUSIONS: Recruitment of slightly more than the minimal target number of end-of-life cancer patients in this study in general practice was realized. Monitoring of all eligible patients permitted to evaluate the selection which occurred. Recruitment through GPs who were direct professional colleagues of one of the researchers was a positive recruitment factor.

Combination of a spinning disc confocal unit with frequency-domain fluorescence lifetime imaging microscopy.

BACKGROUND: Wide-field frequency-domain fluorescence lifetime imaging microscopy (FLIM) is an established technique to determine fluorescence lifetimes. Disadvantage of wide-field imaging is that measurements are compromised by out-of-focus blur. Conventional scanning confocal typically means long acquisition times and more photo bleaching. An alternative is spinning-disc confocal whereby samples are scanned simultaneously by thousands of pinholes, resulting in a virtually instantaneous image with more than tenfold reduced photo bleaching. METHODS: A spinning disc unit was integrated into an existing FLIM system. Measurements were made of fluorescent beads with a lifetime of 2.2 ns against a 5.3 ns fluorescent background outside the focal plane. In addition, living HeLa cells were imaged with different lifetimes in the cytosol and the plasma membrane. RESULTS: In spinning-disc mode, a lifetime of the beads of 2.8 ns was measured, whereas in wide field a lifetime of 4.1 ns was measured. Lifetime contrast within living HeLa cells could be resolved with the spinning-disc unit, where this was impossible in wide field. CONCLUSIONS: Integration of a spinning-disc unit into a frequency-domain FLIM instrument considerably reduces artifacts, while maintaining the advantages of wide field. For FLIM on objects with 3D lifetime structure, spinning-disc is by far preferable over wide-field measurements.

In vivo fluorescence correlation microscopy (FCM) reveals accumulation and immobilization of Nod factors in root hair cell walls.

Fluorescence correlation microscopy (FCM) is a new single-molecule detection technique based on the confocal principle to quantify molecular diffusion and concentration of fluorescent molecules (particles) with sub-micron resolution. In this study, FCM is applied to examine the diffusional behaviour of fluorescent Nod factor analogues on living Vicia sativa root hairs. Three recently described Nod factors with a fluorescent acyl chain (Goedhart et al. Biochemistry 1999, 38, 10898-10907) were used. Plasmolysis of fluorescently labelled root hairs showed that the Nod factors are predominantly located in the cell wall, as hardly any fluorescence could be detected in the plasma membrane. After Nod factor-induced root hair deformation, the new outgrowth was not labelled, indicating a lack of migration of Nod factors to the newly synthesized cell wall. In agreement, FCM showed a > 1,000-fold reduction of molecular mobility of the fluorescence Nod factors upon binding to the cell wall. In addition, FCM demonstrated that Nod factors, when exogenously applied in aqueous solution at 10 nM, markedly concentrate in the cell wall of root hairs (up to 50-fold). The feasibility of applying FCM for the study of living plant cells as well as the implications of our results for the perception of Nod factors are discussed.

Nod factors integrate spontaneously in biomembranes and transfer rapidly between membranes and to root hairs, but transbilayer flip-flop does not occur.

Three novel nodulation (Nod) factors were synthesized from chitotetraose and three structurally different fluorescent BODIPY-tagged fatty acids. With fluorescence spectroscopic and microscopic techniques, the following aspects were studied: whether these amphiphilic molecules insert in membranes, whether they transfer between different membranes, and whether they are able to transfer from a membrane to a legume root hair. Fluorescence correlation spectroscopy showed that fluorescent Nod factors are present as monomers in PBS buffer at a concentration of 10 nM, but that when either Triton X-100 micelles or dioleoylphosphatidylcholine (DOPC) vesicles are present, the Nod factors are associated with these particles. With time-correlated single-photon counting fluorescence spectroscopy, it was shown that upon Nod factor insertion in the membrane, the rotation of the fluorescent acyl chain was markedly reduced. A fluorescence resonance energy transfer assay was used to study the transfer of Nod factors from one membrane to the other, or from vesicles to root hairs. Nod factors transfer rapidly between membranes or from vesicles to root hair cell walls. However, they do not flip-flop between membrane leaflets. The results provide novel insights for the mode of secretion and transfer of Nod factors during the early steps of the Rhizobium-legume interaction.