Limits...
Accessible Synthetic Probes for Staining Actin inside Platelets and Megakaryocytes by Employing Lifeact Peptide.

Cardo L, Thomas SG, Mazharian A, Pikramenou Z, Rappoport JZ, Hannon MJ, Watson SP - Chembiochem (2015)

Bottom Line: Lifeact is a 17-residue peptide that can be employed in cell microscopy as a probe for F-actin when fused to fluorescent proteins, but therefore is not suitable for all cell types.Using confocal microscopy, we characterised the cell distribution of the new hybrids in fixed cells, and found that both myr- and pHLIP-Lifeact conjugates provide efficient actin staining upon cleavage of Lifeact from the carriers, without affecting cell spreading.This new approach could facilitate the design of new tools for actin visualisation.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT (UK).

No MeSH data available.


Related in: MedlinePlus

Actin staining in MKs. MKs were incubated with 5 μm of A) 5 or B) 6 spread on fibrinogen, fixed and analysed by confocal fluorescence microscopy. Lifeact(FAM), carrier(TAMRA) and FRET images are on the left, middle and right, respectively. Scale bars: 5 μm. A typical podosome structure is indicated with a white arrow in (A). C) Mean emission intensities (±SEM) of Lifeact(FAM), carrier(TAMRA) and FRET with 5 (▪) and 6 (▪); 20 cells from two different experiments were used for each measurement.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4524417&req=5

fig04: Actin staining in MKs. MKs were incubated with 5 μm of A) 5 or B) 6 spread on fibrinogen, fixed and analysed by confocal fluorescence microscopy. Lifeact(FAM), carrier(TAMRA) and FRET images are on the left, middle and right, respectively. Scale bars: 5 μm. A typical podosome structure is indicated with a white arrow in (A). C) Mean emission intensities (±SEM) of Lifeact(FAM), carrier(TAMRA) and FRET with 5 (▪) and 6 (▪); 20 cells from two different experiments were used for each measurement.

Mentions: Since 5 and 6 are promising actin markers for platelets, we tested their efficiency in megakaryocytes (MKs), which are responsible for platelet production. Figure 4 A and B shows two representative examples of MKs pretreated with 5 and 6, respectively, spread on fibrinogen and fixed. Actin staining was observed for compounds (Lifact(FAM) emission images on the left), including marking of typical podosome structures (indicated in the Figure 4 A).[22] As observed in platelets, myristoylated carrier is densely accumulated in cells (carrier(TAMRA) emission images in the middle), whereas the emission of pHLIP is significantly lower. Due to the highest complexity of actin filament organisation in these cells, colocalisation between carriers and Lifeact is complex, and FRET microscopy is important for interpretation: the images on the right were obtained by detecting TAMRA emission caused by energy transfer upon excitation of FAM at 488 nm; this occurs only if Lifeact is anchored to the carrier. FRET intensity was higher than FAM emission intensity in cells treated with 5 (Figure 4 A), thus indicating a large amount of uncleaved compound in cells. Instead, only low FRET was detected in MKs treated with 6 (Figure 4 B), thus indicating low accumulation of uncleaved conjugate (see analysis in Figure 4 C).


Accessible Synthetic Probes for Staining Actin inside Platelets and Megakaryocytes by Employing Lifeact Peptide.

Cardo L, Thomas SG, Mazharian A, Pikramenou Z, Rappoport JZ, Hannon MJ, Watson SP - Chembiochem (2015)

Actin staining in MKs. MKs were incubated with 5 μm of A) 5 or B) 6 spread on fibrinogen, fixed and analysed by confocal fluorescence microscopy. Lifeact(FAM), carrier(TAMRA) and FRET images are on the left, middle and right, respectively. Scale bars: 5 μm. A typical podosome structure is indicated with a white arrow in (A). C) Mean emission intensities (±SEM) of Lifeact(FAM), carrier(TAMRA) and FRET with 5 (▪) and 6 (▪); 20 cells from two different experiments were used for each measurement.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4524417&req=5

fig04: Actin staining in MKs. MKs were incubated with 5 μm of A) 5 or B) 6 spread on fibrinogen, fixed and analysed by confocal fluorescence microscopy. Lifeact(FAM), carrier(TAMRA) and FRET images are on the left, middle and right, respectively. Scale bars: 5 μm. A typical podosome structure is indicated with a white arrow in (A). C) Mean emission intensities (±SEM) of Lifeact(FAM), carrier(TAMRA) and FRET with 5 (▪) and 6 (▪); 20 cells from two different experiments were used for each measurement.
Mentions: Since 5 and 6 are promising actin markers for platelets, we tested their efficiency in megakaryocytes (MKs), which are responsible for platelet production. Figure 4 A and B shows two representative examples of MKs pretreated with 5 and 6, respectively, spread on fibrinogen and fixed. Actin staining was observed for compounds (Lifact(FAM) emission images on the left), including marking of typical podosome structures (indicated in the Figure 4 A).[22] As observed in platelets, myristoylated carrier is densely accumulated in cells (carrier(TAMRA) emission images in the middle), whereas the emission of pHLIP is significantly lower. Due to the highest complexity of actin filament organisation in these cells, colocalisation between carriers and Lifeact is complex, and FRET microscopy is important for interpretation: the images on the right were obtained by detecting TAMRA emission caused by energy transfer upon excitation of FAM at 488 nm; this occurs only if Lifeact is anchored to the carrier. FRET intensity was higher than FAM emission intensity in cells treated with 5 (Figure 4 A), thus indicating a large amount of uncleaved compound in cells. Instead, only low FRET was detected in MKs treated with 6 (Figure 4 B), thus indicating low accumulation of uncleaved conjugate (see analysis in Figure 4 C).

Bottom Line: Lifeact is a 17-residue peptide that can be employed in cell microscopy as a probe for F-actin when fused to fluorescent proteins, but therefore is not suitable for all cell types.Using confocal microscopy, we characterised the cell distribution of the new hybrids in fixed cells, and found that both myr- and pHLIP-Lifeact conjugates provide efficient actin staining upon cleavage of Lifeact from the carriers, without affecting cell spreading.This new approach could facilitate the design of new tools for actin visualisation.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT (UK).

No MeSH data available.


Related in: MedlinePlus