scholarly journals Microtubules can bear enhanced compressive loads in living cells because of lateral reinforcement

2006 ◽  
Vol 173 (5) ◽  
pp. 733-741 ◽  
Author(s):  
Clifford P. Brangwynne ◽  
Frederick C. MacKintosh ◽  
Sanjay Kumar ◽  
Nicholas A. Geisse ◽  
Jennifer Talbot ◽  
...  
Keyword(s):  
Cell Shape ◽  
Large Scale ◽  
Living Cells ◽  
Mechanical Coupling ◽  
Compressive Loads ◽  
Structural Contribution ◽  
Cytoplasmic Microtubules ◽  
Lateral Reinforcement

Cytoskeletal microtubules have been proposed to influence cell shape and mechanics based on their ability to resist large-scale compressive forces exerted by the surrounding contractile cytoskeleton. Consistent with this, cytoplasmic microtubules are often highly curved and appear buckled because of compressive loads. However, the results of in vitro studies suggest that microtubules should buckle at much larger length scales, withstanding only exceedingly small compressive forces. This discrepancy calls into question the structural role of microtubules, and highlights our lack of quantitative knowledge of the magnitude of the forces they experience and can withstand in living cells. We show that intracellular microtubules do bear large-scale compressive loads from a variety of physiological forces, but their buckling wavelength is reduced significantly because of mechanical coupling to the surrounding elastic cytoskeleton. We quantitatively explain this behavior, and show that this coupling dramatically increases the compressive forces that microtubules can sustain, suggesting they can make a more significant structural contribution to the mechanical behavior of the cell than previously thought possible.

scholarly journals THE ROLE OF ALKALINE PHOSPHATASE IN OSTEOGENESIS

1951 ◽  
Vol 93 (5) ◽  
pp. 415-426 ◽  
Author(s):  
Robert S. Siffert
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Bone Matrix ◽  
Living Cells ◽  
Alkaline Solutions ◽  
Human Beings ◽  
Acid Media ◽  
Weakly Acid

The role of alkaline phosphatase in osteogenesis has been investigated by histochemical techniques with particular attention to its relationship to phosphate metabolism and matrix elaboration. The upper tibial epiphysis mainly, and other epiphyses as well of growing rabbits, and the costochondral junctions of newborn human beings were studied, as were bone grafts in growing rabbits. The findings in the newborn human beings were identical with those in the rabbits. Phosphatase activity and free phosphate localization do not universally coincide. The enzyme appears to be intimately related to preosseous cellular metabolism and to the elaboration of a bone matrix that is chemically calcifiable. It remains possible, however, that phosphatase may be in some way involved in making inorganic salts available to the calcifiable matrix. If this function does exist it is a secondary one, since the elaboration of bone matrix, which is always associated with phosphatase activity, can and does occur in the absence of calcification. Calcification may occur later, in the absence of the enzyme. There is evidence to suggest that cartilage matrix is utilized in the formation of bone matrix. Phosphatase is physiologically active only in the presence of living cells. Where it is demonstrable in the absence of living cells, as in the cartilage remnants of the metaphysis, it appears to be physiologically inactive. Since phosphatase is temporarily inactivated in weakly acid media, and readily reactivated by alkaline solutions it is possible that the enzyme might survive in a physiologically inactive state in weakly acid tissues, and yet remain capable of histochemical demonstration in vitro in an alkaline medium. Phosphatase is not related to the disappearance of chondroitin sulfate.

An Actin-Like Component in Sperm Tails of A Crane Fly (Nephrotoma Suturalis Loew)

1972 ◽  
Vol 11 (2) ◽  
pp. 491-519
Author(s):  
A. FORER ◽  
O. BEHNKE
Keyword(s):  
Small Groups ◽  
Living Cells ◽  
Mature Sperm ◽  
Heavy Meromyosin ◽  
Cytoplasmic Microtubules ◽  
Nuclear Regions ◽  
To Come ◽  
Oriented Parallel

In negatively stained preparations made from glycerinated crane fly spermatids and sperm, actin-like filaments are seen which bind heavy meromyosin (HMM) to form arrowhead complexes, the reaction with HMM being blocked by ATP and pyrophosphate. In preparations from young spermatids the actin-like filaments are found singly, or in small groups, while in those from mature sperm the actin-like filaments are organized into a structure which we call ‘rods’. Both rods and single filaments come from lysed sperm tails. The actin-like filaments in rods bind HMM only when frayed out on a grid. In sections of normal or glycerinated spermatids or sperm, no actin-like filaments are seen, either because they are not preserved through the fixation and embedding procedures, or because they are present in a form which we do not recognize. In sections of glycerinated spermatids incubated with HMM, decorated filaments are seen in non-nuclear regions of spermatids (tails), oriented parallel to the axoneme. These probably correspond to the single filaments identified in the negatively stained preparations and not to the rods, because the actin-like filaments in rods bind HMM only after fraying out on a grid. HMM causes polymerization of filaments: actin-like filaments are not seen in negatively stained preparations of glycerinated cells subsequently incubated in salts solution, yet are seen in such cells after a further incubation in HMM. Thus some of the decorated filaments seen in sections may have been polymerized by the HMM, raising questions about the procedure. After rupture of living cells no single actin-like filaments are seen in negatively stained preparations, raising the question of whether glycerol as well as HMM can cause polymerization of filaments. Rods are seen in such preparations, and thus rods seem to exist as such in living cells. Single actin-like filaments and rods are seen even when cytoplasmic microtubules are not seen (after incubation in colchicine or vinblastine). Thus the filaments do not seem to come directly from cytoplasmic microtubules. The possible presence of actin-like filaments in cilia and in other flagella, the location of actin-like filaments in sperm tails, and the possible role of the actin-like filaments are discussed.

Force-dependent vinculin binding to talin in live cells: a crucial step in anchoring the actin cytoskeleton to focal adhesions

2014 ◽  
Vol 306 (6) ◽  
pp. C607-C620 ◽  
Author(s):  
Hiroaki Hirata ◽  
Hitoshi Tatsumi ◽  
Chwee Teck Lim ◽  
Masahiro Sokabe
Keyword(s):  
Actin Cytoskeleton ◽  
Focal Adhesions ◽  
Living Cells ◽  
Live Cells ◽  
Mechanical Forces ◽  
Actin Network ◽  
Crucial Step

Mechanical forces play a pivotal role in the regulation of focal adhesions (FAs) where the actin cytoskeleton is anchored to the extracellular matrix through integrin and a variety of linker proteins including talin and vinculin. The localization of vinculin at FAs depends on mechanical forces. While in vitro studies have demonstrated the force-induced increase in vinculin binding to talin, it remains unclear whether such a mechanism exists at FAs in vivo. In this study, using fibroblasts cultured on elastic silicone substrata, we have examined the role of forces in modulating talin-vinculin binding at FAs. Stretching the substrata caused vinculin accumulation at talin-containing FAs, and this accumulation was abrogated by expressing the talin-binding domain of vinculin (domain D1, which inhibits endogenous vinculin from binding to talin). These results indicate that mechanical forces loaded to FAs facilitate vinculin binding to talin at FAs. In cell-protruding regions, the actin network moved backward over talin-containing FAs in domain D1-expressing cells while it was anchored to FAs in control cells, suggesting that the force-dependent vinculin binding to talin is crucial for anchoring the actin cytoskeleton to FAs in living cells.

scholarly journals Visualization of Periplasmic and Cytoplasmic Proteins with a Self-Labeling Protein Tag

2016 ◽  
Vol 198 (7) ◽  
pp. 1035-1043 ◽  
Author(s):  
Na Ke ◽  
Dirk Landgraf ◽  
Johan Paulsson ◽  
Mehmet Berkmen
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Functional Protein ◽  
Content Type ◽  
Additional Tool

ABSTRACTThe use of fluorescent and luminescent proteins in visualizing proteins has become a powerful tool in understanding molecular and cellular processes within living organisms. This success has resulted in an ever-increasing demand for new and more versatile protein-labeling tools that permit light-based detection of proteins within living cells. In this report, we present data supporting the use of the self-labeling HaloTag protein as a light-emitting reporter for protein fusions within the model prokaryoteEscherichia coli. We show that functional protein fusions of the HaloTag can be detected bothin vivoandin vitrowhen expressed within the cytoplasmic or periplasmic compartments ofE. coli. The capacity to visually detect proteins localized in various prokaryotic compartments expands today's molecular biologist toolbox and paves the path to new applications.IMPORTANCEVisualizing proteins microscopically within living cells is important for understanding both the biology of cells and the role of proteins within living cells. Currently, the most common tool is green fluorescent protein (GFP). However, fluorescent proteins such as GFP have many limitations; therefore, the field of molecular biology is always in need of new tools to visualize proteins. In this paper, we demonstrate, for the first time, the use of HaloTag to visualize proteins in two different compartments within the model prokaryoteEscherichia coli. The use of HaloTag as an additional tool to visualize proteins within prokaryotes increases our capacity to ask about and understand the role of proteins within living cells.

scholarly journals ERK Activity Imaging During Migration of Living Cells In Vitro and In Vivo

2019 ◽  
Vol 20 (3) ◽  
pp. 679 ◽  
Author(s):  
Eishu Hirata ◽  
Etsuko Kiyokawa
Keyword(s):  
Cancer Metastasis ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Cell Dynamics ◽  
Extracellular Signal

Extracellular signal-regulated kinase (ERK) is a major downstream factor of the EGFR-RAS-RAF signalling pathway, and thus the role of ERK in cell growth has been widely examined. The development of biosensors based on fluorescent proteins has enabled us to measure ERK activities in living cells, both after growth factor stimulation and in its absence. Long-term imaging unexpectedly revealed the oscillative activation of ERK in an epithelial sheet or a cyst in vitro. Studies using transgenic mice expressing the ERK biosensor have revealed inhomogeneous ERK activities among various cell species. In vivo Förster (or fluorescence) resonance energy transfer (FRET) imaging shed light on a novel role of ERK in cell migration. Neutrophils and epithelial cells in various organs such as intestine, skin, lung and bladder showed spatio-temporally different cell dynamics and ERK activities. Experiments using inhibitors confirmed that ERK activities are required for various pathological responses, including epithelial repair after injuries, inflammation, and niche formation of cancer metastasis. In conclusion, biosensors for ERK will be powerful and valuable tools to investigate the roles of ERK in situ.

scholarly journals Acquiring Metastatic Competence by Oral Squamous Cell Carcinoma Cells Is Associated with Differential Expression ofα-Tubulin Isoforms

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Becky Lou ◽  
David Engler ◽  
William Dubinsky ◽  
Jean Wu ◽  
Nadarajah Vigneswaran
Keyword(s):  
Cell Lines ◽  
Cell Shape ◽  
Quantitative Difference ◽  
Distribution Patterns ◽  
Carcinoma Cells ◽  
Protein Levels ◽  
Distinct Cell ◽  
Tubulin Isoforms

We performed comparative global proteomics analyses of patient-matched primary (686Tu) and metastatic (686Ln) OSCC cells. The metastatic OSCC 686Ln cells showed greaterin vitromigratory/invasive potential and distinct cell shape from their parental primary 686Tu cells. Ettan DIGE analysis revealed 1316 proteins spots in both cell lines with >85% to be quantitatively similar (<2 folds) between the two cell lines. However, two protein spots among four serial spots were highly dominant in 686Ln cells. Mass spectrometry sequencing demonstrated all four spots to beα-tubulin isotypes. Further analysis showed no significant quantitative difference in theα-tubulin between the two cell lines either at mRNA or protein levels. Thus, two distinct isoforms ofα-tubulin, probably due to posttranslational modification, were associated with metastatic 686Ln cells. Immunofluorescence demonstrated remarkable differences in the cytosolicα-tubulin distribution patterns between the two cells. In 686Tu cells,α-tubulin proteins formed a normal network composed of filaments. In contrast,α-tubulin in 686Ln cells exhibited only partial cytoskeletal distribution with the majority of the protein diffusely distributed within the cytosol. Sinceα-tubulin is critical for cell shape and mobility, our finding suggests a role ofα-tubulin isoforms in acquisition of metastatic phenotype and represents potential target for therapeutic intervention.

Differentiation of Secondary Xylem After Girdling

IAWA Journal ◽  
1988 ◽  
Vol 9 (4) ◽  
pp. 375-383 ◽  
Author(s):  
Li Zhengli ◽  
Cui Keming
Keyword(s):  
Large Scale ◽  
Vascular Tissue ◽  
Secondary Xylem ◽  
Living Cells ◽  
Growth Season ◽  
Meristematic Tissue ◽  
Continuous Growth ◽  
Parenchyma Cells

Under favourable growth season and by suitable technical means, regeneration and continuous growth of new bark after girdling has been observed in many trees. Differentiation of the secondary xylem varies after arteficial treatment. Thus , the authors consider that (1) under appropriate conditions most trees could be girdled on a large scale with subsequent new bark regeneration and continued growth, (2) after removal of the phloem the living cells of the secondary xylem, i.e., wood parenchyma cells, may function in transporting nutrients from the treecrown downwards, and (3) finally, after girdling or when cultured in vitro, both immature xylem and phloem can dedifferentiate into meristematic tissue that further develops vascular tissue.

The zoospore of Rozella allomycis: ultrastructure

1975 ◽  
Vol 53 (19) ◽  
pp. 2212-2232 ◽  
Author(s):  
Abraham A. Held
Keyword(s):  
Cell Shape ◽  
Bilateral Symmetry ◽  
Basic Pattern ◽  
Structural Peculiarities ◽  
Olpidium Brassicae ◽  
Cytoplasmic Microtubules ◽  
Cytoskeletal Elements

The small (about 2 × 3.5 μm) zoospore of the obligately parasitic chytrid Rozella allomycis provides yet another ultrastructural variation on the basic pattern of posteriorly uniflagellate fungal zoospores. This zoospore contains the expected array of organelles: nucleus, mitochondria, "lipid sac" (including lipid globules, a microbody, and a backing membrane), kinetosome, and second centriole. Ribosomes are dispersed throughout the cytoplasm. In the disposition of its microbody Rozella resembles other chytrids, whereas in the occurrence and location of its gamma-like vacuoles it resembles certain blastocladialean fungi. Above all, the zoospore resembles that of Olpidium brassicae. Structural peculiarities of the zoospore include (i) bilateral symmetry; (ii) a helmet-shaped nucleus with extensive membranous projections which surmounts a large, spheroidal mitochondrion; and (iii) a particularly elongate cell shape because of the unusual length (about 1 μm) of the kinetosome and the presence of a 1-μm-long flagellar cavity which surrounds the base of the flagellum. Cytoskeletal elements include cytoplasmic microtubules, microfilaments which mainly reinforce the flagellar cavity, and props which anchor the kinetosome in the plasmalemma at the roof of the flagellar cavity. The role of these elements is discussed. A revision of the description of the olpidiaceous zoospore is suggested.

Sign in / Sign up

Export Citation Format

Share Document