Pronounced cytoplasmic pH gradients are not required for tip growth in plant and fungal cells

1997 ◽  
Vol 110 (10) ◽  
pp. 1187-1198 ◽  
Author(s):  
R.M. Parton ◽  
S. Fischer ◽  
R. Malho ◽  
O. Papasouliotis ◽  
T.C. Jelitto ◽  
...  
Keyword(s):  
Tip Growth ◽  
Cell Types ◽  
Pollen Tubes ◽  
Fine Tuning ◽  
Cytoplasmic Ph ◽  
Dual Emission ◽  
Ph Gradients ◽  
Longitudinal Gradients ◽  
External Ph

The existence of pronounced cytoplasmic pH gradients within the apices of tip-growing cells, and the role of cytoplasmic pH in regulating tip growth, were investigated in three different cell types: vegetative hyphae of Neurospora crassa; pollen tubes of Agapanthus umbellatus; and rhizoids of Dryopteris affinis gametophytes. Examination of cytoplasmic pH in growing cells was performed by simultaneous, dual emission confocal ratio imaging of the pH-sensitive probe carboxy SNARF-1. Considerable attention was paid to the fine tuning of dye loading and imaging parameters to minimise cellular perturbation and assess the extent of dye partitioning into organelles. With optimal conditions, cytoplasmic pH was measured routinely with a precision of between +/−0.03 and +/−0.06 of a pH unit and a spatial resolution of 2.3 microm2. Based on in vitro calibration, estimated values of mean cytoplasmic pH for cells loaded with dye-ester were between 7.15 and 7.25 for the three cell types. After pressure injecting Neurospora hyphae with dextran-conjugated dye, however, the mean cytoplasmic pH was estimated to be 7.57. Dextran dyes are believed to give a better estimate of cytoplasmic pH because of their superior localisation and retention within the cytosol. No significant cytoplasmic pH gradient (delta pH of >0.1 unit) was observed within the apical 50 microm in growing cells of any of the three cell types. Acidification or alkalinisation of the cytoplasm in Neurospora hyphae, using a cell permeant weak acid (propionic acid at pH 7.0) or weak base (trimethylamine at pH 8.0), slowed down but did not abolish growth. However, similar manipulation of the cytoplasmic pH of Agapanthus pollen tubes and Dryopteris rhizoids completely inhibited growth. Modification of external pH affected the growth pattern of all cell types. In hyphae and pollen tubes, changes in external pH were found to have a small transient effect on cytoplasmic pH but the cells rapidly readjusted towards their original pH. Our results suggest that pronounced longitudinal gradients in cytoplasmic pH are not essential for the regulation of tip growth.

scholarly journals pH gradients are not associated with tip growth in pollen tubes of Lilium longiflorum

1997 ◽  
Vol 110 (15) ◽  
pp. 1729-1740 ◽  
Author(s):  
M.D. Fricker ◽  
N.S. White ◽  
G. Obermeyer
Keyword(s):  
Pollen Tube ◽  
Tip Growth ◽  
Lilium Longiflorum ◽  
Pollen Tubes ◽  
Tube Growth ◽  
Cytoplasmic Ph ◽  
Clear Zone ◽  
External Application ◽  
Ph Gradients ◽  
External Ph

The cytoplasmic pH of growing pollen tubes of Lilium longiflorum Thunb. was measured using the pH-sensitive fluorescent dye 2′,7′-bis-(carboxyethyl)-5(6′)-carboxyfl uorescein and confocal fluorescence ratio imaging. The average cytoplasmic pH in the clear zone of the pollen tube tip was pH 7.11, and no consistent pH gradients were detected in the clear zone, averaging around -1.00 milli pH unit microm(−1), or along the first 50 microm of the tube (3.62 milli pH units microm[-1]). In addition, no correlation was observed between the absolute tip cytoplasmic pH or the pH gradient and the pollen tube growth rates. Shifts of external pH to more acidic pH values (pH 4.5) caused a relatively small acidification by 0.18 pH units, whereas a more alkaline external pH >7.0 caused a dramatic increase in cytoplasmic pH and growth stopped immediately. Stimulation of the plasma membrane H+-ATPase by fusicoccin, resulted in an increase of tube growth but no change in cytoplasmic pH. On the other hand, vanadate (250–500 microM), a putative inhibitor of the pump, stopped tube growth and a slight cytoplasmic alkalinisation of 0.1 pH units was observed. Vanadate also arrested fusicoccin-stimulated growth and stimulated an increased alkalinisation of around 0.2 pH units. External application of CaCl2 (10 mM) caused a small acidification of less than 0.1 pH units in the clear zone, whilst LaCl3 (250 microM) caused slight and rather variable perturbations in cytoplasmic pH of no more than 0.1 pH units. Both treatments stopped growth. It was inferred from these data that tip-acid cytoplasmic pH gradients do not play a central role in the organisation or maintenance of pollen tube tip growth.

scholarly journals Protein Analysis of Pollen Tubes after the Treatments of Membrane Trafficking Inhibitors Gains Insights on Molecular Mechanism Underlying Pollen Tube Polar Growth

2021 ◽  
Vol 40 (2) ◽  
pp. 205-222
Author(s):  
Monica Scali ◽  
Alessandra Moscatelli ◽  
Luca Bini ◽  
Elisabetta Onelli ◽  
Rita Vignani ◽  
...  
Keyword(s):  
Pollen Tube ◽  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Tip Growth ◽  
Male Gametophyte ◽  
Pollen Tubes ◽  
Structural Features ◽  
Two Dimensional Gel Electrophoresis ◽  
Depth Analysis

AbstractPollen tube elongation is characterized by a highly-polarized tip growth process dependent on an efficient vesicular transport system and largely mobilized by actin cytoskeleton. Pollen tubes are an ideal model system to study exocytosis, endocytosis, membrane recycling, and signaling network coordinating cellular processes, structural organization and vesicular trafficking activities required for tip growth. Proteomic analysis was applied to identifyNicotiana tabacumDifferentially Abundant Proteins (DAPs) after in vitro pollen tube treatment with membrane trafficking inhibitors Brefeldin A, Ikarugamycin and Wortmannin. Among roughly 360 proteins separated in two-dimensional gel electrophoresis, a total of 40 spots visibly changing between treated and control samples were identified by MALDI-TOF MS and LC–ESI–MS/MS analysis. The identified proteins were classified according to biological processes, and most proteins were related to pollen tube energy metabolism, including ammino acid synthesis and lipid metabolism, structural features of pollen tube growth as well modification and actin cytoskeleton organization, stress response, and protein degradation. In-depth analysis of proteins corresponding to energy-related pathways revealed the male gametophyte to be a reliable model of energy reservoir and dynamics.

Morphology of Cell Injury: An Approach to the EDIT Programme by the Use of Tobacco Pollen Tubes

2002 ◽  
Vol 30 (3) ◽  
pp. 323-329 ◽  
Author(s):  
Udo Kristen ◽  
Natalie Bischoff ◽  
Saskia Lisboa ◽  
Enno Schirmer ◽  
Sören Witt ◽  
...  
Keyword(s):  
Tip Growth ◽  
Cell Injury ◽  
Cytosolic Calcium ◽  
Pollen Tubes ◽  
In Vitro Cytotoxicity ◽  
Toxic Compounds ◽  
In Vitro System ◽  
Tobacco Pollen ◽  
Different Types

Tobacco pollen tubes were used as a standard in vitro system to investigate cell growth aberrations caused by some of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme chemicals and other toxic compounds. Changes in cytoskeletal pattern were observed in the tube cells by using tubu-lin immunofluorescence and rhodamin–phalloidin fluorescence for the localisation of microtubules and actin filaments, respectively. Four different types of cell malformation were found: screw-like growth, isodiametric tip swelling, hook formation, and pollen grain enlargement. We suggest that these malformations resulted from an interference by the chemicals with the cytosolic calcium gradient which controls tip growth and the orientation of the pollen tube. The results may contribute to a general understanding of toxicity-based cell malformations.

Cytosolic pH measurements in single cardiac myocytes using carboxy-seminaphthorhodafluor-1

1992 ◽  
Vol 263 (1) ◽  
pp. H276-H284 ◽  
Author(s):  
P. S. Blank ◽  
H. S. Silverman ◽  
O. Y. Chung ◽  
B. A. Hogue ◽  
M. D. Stern ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Spectral Feature ◽  
Ph Measurements ◽  
Dual Emission ◽  
Cytosolic Ph ◽  
Ph Gradients ◽  
In Situ Calibration ◽  
Rat Cardiac Myocytes

This study examines the use of carboxy-seminaphthorhodafluor-1 (C-SNARF-1) as an indicator of cytosolic pH in isolated rat cardiac myocytes. The emission spectrum of C-SNARF-1 when excited at 530 nm contains two well-separated peaks at approximately 590 and 640 nm, corresponding to the acidic and basic forms of the indicator. This spectral feature allows the indicator to be used in the single excitation, dual emission ratio mode. When C-SNARF-1 is loaded into rat cardiac myocytes as the membrane permeant ester derivative, C-SNARF-1/AM, the indicator localizes within the cytosol with virtually no partitioning into the mitochondria. C-SNARF-1 does not load into isolated mitochondria in suspension. There was no evidence for the presence of non-deesterified C-SNARF-1 within the cells. C-SNARF-1 can be calibrated in situ using a technique that abolishes all transsarcolemmal pH gradients. A 0.7-unit shift in the apparent pK (pKapp = pK-log10) between the in vitro calibration and the in situ calibration is consistent with a change in beta (I640 to pH 9/I640 at pH 5) in the cytosolic environment (beta in situ/beta in vitro = 0.21) and not a change in the true pK of the indicator. The contribution of cellular autofluorescence to the total signal can be made negligible. There is no effect of C-SNARF-1 on the contractile properties of rat cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Estimation of cellular pH gradients with 31P-NMR in intact rabbit renal tubular cells

1984 ◽  
Vol 247 (3) ◽  
pp. C188-C196 ◽  
Author(s):  
S. Adler ◽  
E. Shoubridge ◽  
G. K. Radda
Keyword(s):  
Ph Gradient ◽  
Cytoplasmic Ph ◽  
Buffer Solution ◽  
Ph Gradients ◽  
Tubular Cells ◽  
Renal Tubular Cells ◽  
Ph Values ◽  
Intact Rabbit ◽  
Renal Tubular ◽  
External Ph

To determine intracellular pH gradients rabbit renal cortical tubular cells were prepared by collagenase separation, suspended in a Krebs-Ringer buffer solution, and gassed with 95% O2-5% CO2 in a special nuclear magnetic resonance (NMR) probe. Renal tubular cellular pH was determined simultaneously from the distribution of 14C-dimethadione (DMO) (pHDMO) or the chemical shift of inorganic phosphate (pHNMR). Experiments were performed at different external pH values (pHe) ranging between 6.52 and 7.20. pHNMR, a measure of cytoplasmic pH, changed by an amount equal to the change in pHe. pHDMO, however, a measure of cytoplasmic plus mitochondrial pH, changed less than pHe as the latter increased. pHDMO, higher than pHNMR at low pHe, became equal to pHNMR at higher pHe values. By use of assumed mitochondrial volumes of 30-40% mitochondrial pH was calculated from pHDMO and pHNMR. Mitochondrial pH remained relatively constant over the entire pHe range studied. Since cytoplasmic pH fell as pHe was lowered, the transmitochondrial pH gradient increased at low pHE values. These findings suggest that the transmitochondrial pH gradient may be important in regulating metabolism.

scholarly journals Exocytosis and endocytosis: coordinating and fine-tuning the polar tip growth domain in pollen tubes

2020 ◽  
Vol 71 (8) ◽  
pp. 2428-2438 ◽  
Author(s):  
Jingzhe Guo ◽  
Zhenbiao Yang
Keyword(s):  
Cell Wall ◽  
Rho Gtpases ◽  
Intracellular Signaling ◽  
Tip Growth ◽  
Apical Cell ◽  
Turgor Pressure ◽  
Pollen Tubes ◽  
Fine Tuning ◽  
Rop Gtpase ◽  
Specialized Form

Abstract Pollen tubes rapidly elongate, penetrate, and navigate through multiple female tissues to reach ovules for sperm delivery by utilizing a specialized form of polar growth known as tip growth. This process requires a battery of cellular activities differentially occurring at the apical growing region of the plasma membrane (PM), such as the differential cellular signaling involving calcium (Ca2+), phospholipids, and ROP-type Rho GTPases, fluctuation of ions and pH, exocytosis and endocytosis, and cell wall construction and remodeling. There is an emerging understanding of how at least some of these activities are coordinated and/or interconnected. The apical active ROP modulates exocytosis to the cell apex for PM and cell wall expansion differentially occurring at the tip. The differentiation of the cell wall involves at least the preferential distribution of deformable pectin polymers to the apex and non-deformable pectin polymers to the shank of pollen tubes, facilitating the apical cell expansion driven by high internal turgor pressure. Recent studies have generated inroads into how the ROP GTPase-based intracellular signaling is coordinated spatiotemporally with the external wall mechanics to maintain the tubular cell shape and how the apical cell wall mechanics are regulated to allow rapid tip growth while maintaining the cell wall integrity under the turgor pressure. Evidence suggests that exocytosis and endocytosis play crucial but distinct roles in this spatiotemporal coordination. In this review, we summarize recent advances in the regulation and coordination of the differential pectin distribution and the apical domain of active ROP by exocytosis and endocytosis in pollen tubes.

scholarly journals Nifedipine-sensitive calcium channels are involved in polar growth of lily pollen tubes

1985 ◽  
Vol 76 (1) ◽  
pp. 247-254
Author(s):  
H.D. Reiss ◽  
W. Herth
Keyword(s):  
Calcium Channels ◽  
Tip Growth ◽  
Lilium Longiflorum ◽  
Pollen Tubes ◽  
Polar Growth ◽  
Treatment Results ◽  
High Tendency ◽  
Lily Pollen ◽  
Internal Calcium

Pollen germination and tube growth of Lilium longiflorum in vitro are affected by 10(−5) M-nifedipine. Germinating ‘tubes’ form broad protuberances along the whole colpus. Short tubes show a high tendency to grow ‘amoeboid-like’ and to branch; or a second tube emerges in another region of the colpus. Longer tubes (greater than or equal to 100 micron) broaden irregularly or swell at their tips. The diameter of the tube can vary drastically within the same tube. With increasing time of treatment many tubes burst. Normal tip growth stops within 10 min, but protoplasmic streaming continues even after 15 h. More or less regularly, wall thickenings are formed along the whole tube or on the flanks of the germinating region after some hours. The internal calcium gradient, visualized by chlorotetracycline (CTC) fluorescence, is also disturbed. Nifedipine treatment results in uniform or irregular CTC fluorescence. Branching tubes temporarily show a new subapical CTC gradient. After 6–8 h of nifedipine treatment many cells are no longer stainable with CTC. The results indicate the presence of nifedipine-sensitive calcium channels in pollen tubes.

Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

1972 ◽  
Vol 30 ◽  
pp. 350-351
Author(s):  
K. Shankar Narayan ◽  
Kailash C. Gupta ◽  
Tohru Okigaki
Keyword(s):  
Ultraviolet Light ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Survival Rates ◽  
Chinese Hamster ◽  
Cell Types ◽  
Differential Sensitivity ◽  
Ultrastructural Changes ◽  
Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

Ultrastructural observations on the in vitro cytoadherence of Plasmodium falciparum infected red blood cells

1990 ◽  
Vol 48 (3) ◽  
pp. 914-915
Author(s):  
D.J.P. Ferguson ◽  
A.R. Berendt ◽  
J. Tansey ◽  
K. Marsh ◽  
C.I. Newbold
Keyword(s):  
Plasmodium Falciparum ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Umbilical Vein ◽  
Cell Types ◽  
Amelanotic Melanoma ◽  
Cytoplasmic Process ◽  
Umbilical Vein Endothelial Cells

In human malaria, the most serious clinical manifestation is cerebral malaria (CM) due to infection with Plasmodium falciparum. The pathology of CM is thought to relate to the fact that red blood cells containing mature forms of the parasite (PRBC) cytoadhere or sequester to post capillary venules of various tissues including the brain. This in vivo phenomenon has been studied in vitro by examining the cytoadherence of PRBCs to various cell types and purified proteins. To date, three Ijiost receptor molecules have been identified; CD36, ICAM-1 and thrombospondin. The specific changes in the PRBC membrane which mediate cytoadherence are less well understood, but they include the sub-membranous deposition of electron-dense material resulting in surface deformations called knobs. Knobs were thought to be essential for cytoadherence, lput recent work has shown that certain knob-negative (K-) lines can cytoadhere. In the present study, we have used electron microscopy to re-examine the interactions between K+ PRBCs and both C32 amelanotic melanoma cells and human umbilical vein endothelial cells (HUVEC).We confirm previous data demonstrating that C32 cells possess numerous microvilli which adhere to the PRBC, mainly via the knobs (Fig. 1). In contrast, the HUVEC were relatively smooth and the PRBCs appeared partially flattened onto the cell surface (Fig. 2). Furthermore, many of the PRBCs exhibited an invagination of the limiting membrane in the attachment zone, often containing a cytoplasmic process from the endothelial cell (Fig. 2).

Role of the 807 C/T Polymorphism of the α2 Gene in Platelet GP Ia Collagen Receptor Expression and Function

1999 ◽  
Vol 81 (06) ◽  
pp. 951-956 ◽  
Author(s):  
J. Corral ◽  
R. González-Conejero ◽  
J. Rivera ◽  
F. Ortuño ◽  
P. Aparicio ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Cell Types ◽  
Receptor Expression ◽  
Case Control Studies ◽  
Platelet Surface ◽  
Vitro Analysis ◽  
And Function ◽  
In Vitro Analysis

SummaryThe variability of the platelet GP Ia/IIa density has been associated with the 807 C/T polymorphism (Phe 224) of the GP Ia gene in American Caucasian population. We have investigated the genotype and allelic frequencies of this polymorphism in Spanish Caucasians. The T allele was found in 35% of the 284 blood donors analyzed. We confirmed in 159 healthy subjects a significant association between the 807 C/T polymorphism and the platelet GP Ia density. The T allele correlated with high number of GP Ia molecules on platelet surface. In addition, we observed a similar association of this polymorphism with the expression of this protein in other blood cell types. The platelet responsiveness to collagen was determined by “in vitro” analysis of the platelet activation and aggregation response. We found no significant differences in these functional platelet parameters according to the 807 C/T genotype. Finally, results from 3 case/control studies involving 302 consecutive patients (101 with coronary heart disease, 104 with cerebrovascular disease and 97 with deep venous thrombosis) determined that the 807 C/T polymorphism of the GP Ia gene does not represent a risk factor for arterial or venous thrombosis.

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