19 research outputs found
CLSM observations (100X objective) of each FITC-BSA complex using MCTS.
<p>A: Schematic illustration of MCTS observed in these experiments. About one quater size of MCTS was observed from the top view. The optical thickness and the distance of the confocal section from the top of MCTS were 0.38 μm and 20 μm, respectively. B: The intra-MCTS distribution of FITC-BSA (green) complexes with Pro-DeliverIN, Xfect, and TurboFect was observed in detail by CLSM using the 100X objective with staining nuclei (blue). A small amount of FITC-BSA was observed in the nuclei, especially in TurboFect. The scale bars represent 20 μm.</p
CLSM observations (40X objective) of each FITC-BSA complex using MCTS.
<p>A: Schematic illustration of MCTS observed in these experiments. The full size of MCTS was observed from the top view with the Z-stack mode from the top to middle positions. The optical thickness and the distance of the confocal section from the top of MCTS were 0.60 μm and 20 μm, respectively. B: The intra-MCTS distribution of FITC-BSA (green) complexes with Pro-DeliverIN, Xfect, and TurboFect was observed by CLSM using the 40X objective with staining nuclei (blue) shown in A. A certain amount of FITC-BSA complexes with Pro-DeliverIN and Xfect was observed in the inner layered cells. On the other hand, TurboFect showed FITC-BSA distribution only in the surface-first layered cells. The scale bars represent 50 μm.</p
Cellular uptake of each protein transfection reagent/FITC-BSA complex.
<p>FITC-BSA complexes with Pro-DeliverIN, Xfect, and TurboFect were applied to Huh-7 monolayer cultured cells (A) and MCTS (B). Error bars in the graph represent the standard deviation, n = 4. *<i>p</i><0.05 and **<i>p</i><0.01. In the case of monolayer cultured cells (A), TurboFect and Xfect achieved higher cellular uptake efficiency than that of Pro-DeliverIN. MCTS experiments revealed the higher uptake of TurboFect than of Pro-DeliverIN and Xfect. The uptake of protein transfection reagents was markedly higher by monolayer cultured cells than by MCTS.</p
Inhibition of endocytosis using specific inhibitors.
<p>Effects of inhibitors (sucrose: clathrin-mediated endocytosis inhibitor; amiloride: macropinocytosis inhibitor; filipin: caveolae-mediated endocytosis inhibitor) on the internalization of FITC-BSA complexes with Pro-DeliverIN, Xfect, and TurboFect were evaluated using Huh-7 monolayer cultured cells (A) and MCTS (B). Error bars in the graph represent the standard deviation, n = 3 (A) or 4 (B). *<i>p</i><0.05 and **<i>p</i><0.01. The results obtained from monolayer cultured cells (A) and MCTS (B) were similar. Pro-DeliverIN appeared to be internalized into Huh-7 cells by clathrin-mediated endocytosis and caveolae-mediated endocytosis. The uptake route of Xfect was mainly by macropinocytosis, but also slightly by caveolae-mediated endocytosis. TurboFect was preferentially internalized into Huh-7 cells through macropinocytosis. </p
Cell-Penetrating Peptides Using Cyclic α,α-Disubstituted α‑Amino Acids with Basic Functional Groups
In
the delivery of cell-impermeable molecules, cell-penetrating
peptides (CPPs) have been attracting increasing attention as intracellular
delivery tools. In the present study, we designed four types of cyclic
α,α-disubstituted α-amino acids (dAAs) with basic
functional groups on their five-membered rings and different chiralities
at the α-position and introduced them into arginine (Arg)-rich
peptides. The evaluation of cell-penetrating abilities indicated that
these peptides exhibited better cell permeabilities than an Arg nonapeptide.
Furthermore, peptides containing dAAs delivered plasmid DNA (pDNA)
better than a commercially available transfection reagent with a longer
incubation time. These results demonstrate that the introduction of
cyclic dAAs with basic functional groups into Arg-rich peptides is
an effective strategy for the design of CPPs as a pDNA delivery tool
Cell-Penetrating Helical Peptides Having l‑Arginines and Five-Membered Ring α,α-Disubstituted α‑Amino Acids
Cell-penetrating
peptides are powerful tools in the delivery of
drugs, proteins, and nucleic acids into cells; therefore, focus has
recently been placed on their development. In this study, we synthesized
seven types of peptides possessing three l-arginines (l-Arg) and six l-leucines (l-Leu) and/or 1-aminocyclopentane-1-carboxylic
acids (Ac<sub>5</sub>c), and investigated their secondary structures
and cell-penetrating abilities. The peptide composed of an equal number
of l-Arg, l-Leu, and Ac<sub>5</sub>c formed 3<sub>10</sub>/α-helical structures in TFE solution and exhibited
the highest cell-penetrating ability of all the peptides examined.
Additional cellular uptake studies revealed that the incorporation
of Ac<sub>5</sub>c into peptides led to improved tolerability against
serum. The results of the present study will help in the design of
novel cell-penetrating peptides
Rac1-Dependent Lamellipodial Motility in Prostate Cancer PC-3 Cells Revealed by Optogenetic Control of Rac1 Activity
<div><p>The lamellipodium, an essential structure for cell migration, plays an important role in the invasion and metastasis of cancer cells. Although Rac1 recognized as a key player in the formation of lamellipodia, the molecular mechanisms underlying lamellipodial motility are not fully understood. Optogenetic technology enabled us to spatiotemporally control the activity of photoactivatable Rac1 (PA-Rac1) in living cells. Using this system, we revealed the role of phosphatidylinositol 3-kinase (PI3K) in Rac1-dependent lamellipodial motility in PC-3 prostate cancer cells. Through local blue laser irradiation of PA-Rac1-expressing cells, lamellipodial motility was reversibly induced. First, outward extension of a lamellipodium parallel to the substratum was observed. The extended lamellipodium then showed ruffling activity at the periphery. Notably, PI(3,4,5)P<sub>3</sub> and WAVE2 were localized in the extending lamellipodium in a PI3K-dependent manner. We confirmed that the inhibition of PI3K activity greatly suppressed lamellipodial extension, while the ruffling activity was less affected. These results suggest that Rac1-induced lamellipodial motility consists of two distinct activities, PI3K-dependent outward extension and PI3K-independent ruffling.</p></div
Local and reversible control of lamellipodial dynamics by photomanipulation of PA-Rac1 activity.
<p>Time-lapse images of a PC-3 cell expressing mCherry-PA-Rac1 were acquired during PA-Rac1 photo-manipulation by local laser irradiation of different areas. Selected phase-contrast and mCherry fluorescence images are shown. First, region 1 was irradiated for 10 min. The irradiation was then moved to region 2. At 25 min, the irradiation was turned off. Selected time-lapse images of phase-contrast and mCherry fluorescence are shown. The extending and retracting lamellipodia are outlined in red and yellow, respectively. Scale bar, 10 µm.</p
Effect of LY294002 on the extended lamellipodial motility in PC-3 cells expressing constitutively active Rac1Q61L.
<p>PC-3 cells were transfected with pmCitrine-Rac1Q61L. The time-lapse images of phase-contrast and mCitrine-Rac1Q61L fluorescence were captured before (left) and after (right) the addition of LY294002. Kymographs were created to show the changes in length of a lamellipodium at the position of the two-headed line. The mCitrine-Rac1Q61L-expressing cell had an extended lamellipodium around its entire circumference. After the addition of 50 µM LY294002, the extended lamellipodium had shrunk only slightly, but the peripheral ruffling was pronounced. The kymographs show dynamic changes in length due to enhanced ruffling after the addition of LY294002. Scale bars, 10 µm.</p
Enhanced and Prolonged Cell-Penetrating Abilities of Arginine-Rich Peptides by Introducing Cyclic α,α-Disubstituted α‑Amino Acids with Stapling
Cell-penetrating
peptides are receiving increasing attention as
drug delivery tools, and the search for peptides with high cell-penetrating
ability and negligible cytotoxicity has become a critical research
topic. Herein, cyclic α,α-disubstituted α-amino
acids were introduced into arginine-rich peptides and an additional
staple was provided in the side chain. The peptides designed in the
present study showed more enhanced and prolonged cell-penetrating
abilities than an arginine nonapeptide due to high resistance to protease
and conformationally stable helical structures