Assembly and function of Chlamydomonas flagellar mastigonemes as probed with a monoclonal antibody

1996 ◽  
Vol 109 (1) ◽  
pp. 57-62 ◽  
Author(s):  
S. Nakamura ◽  
G. Tanaka ◽  
T. Maeda ◽  
R. Kamiya ◽  
T. Matsunaga ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Beat Frequency ◽  
Distal Portion ◽  
Distal Region ◽  
Ring Structure ◽  
Live Cells ◽  
Swimming Velocity ◽  
Flagellar Beat ◽  
Original Length ◽  
And Function

Mastigonemes are hair-like projections on the flagella of various kinds of lower eukaryotes. We obtained a monoclonal antibody (mAb-MAST1) to mastigonemes of Chlamydomonas reinhardtii, and found that it reacts with a single flagellar glycoprotein of about 230 kDa. Interestingly, immunofluorescence microscopy demonstrated that mAb-MAST1 recognizes not only the flagellar mastigonemes but also a ring composed of 10 or more particles located in the anterior end of the cell body close to the flagellar bases. The ring structure may be the pool of the mastigoneme protein. When the flagella are amputated, they regenerate to their original length in 90–120 minutes. We found that mastigonemes appear on the new flagellar surface as early as 15 minutes after deflagellation, and that new mastigonemes are mostly assembled onto the distal region of the flagellar surface. Mastigonemes thus appear to be inserted into the membrane only in the distal region of the flagellum. Alternatively, mastigonemes may be inserted at the base and transported very rapidly to the distal portion where they are trapped. When live cells are treated with mAb-MAST1, mastigonemes disappear from the flagellar surface. In these mAb-MAST1 treated cells, the swimming velocity decreases to 70–80% of the normal value, although the flagellar beat frequency increases to approximately 110% of the control. These findings demonstrate vectorial transport of mastigonemes to their assembly sites, and show that mastigonemes function to increase flagellar propulsive force by increasing the effective surface of the flagellum.

scholarly journals Effect of beat frequency on the velocity of microtubule sliding in reactivated sea urchin sperm flagella under imposed head vibration.

1995 ◽  
Vol 198 (3) ◽  
pp. 645-653 ◽  
Author(s):  
C Shingyoji ◽  
K Yoshimura ◽  
D Eshel ◽  
K Takahashi ◽  
I R Gibbons
Keyword(s):  
Sea Urchin ◽  
Vibration Frequency ◽  
Beat Frequency ◽  
Distal Region ◽  
Bend Angle ◽  
Vibration Frequencies ◽  
Sliding Velocity ◽  
Flagellar Beat ◽  
Tripneustes Gratilla ◽  
Sea Urchin Sperm

The heads of demembranated spermatozoa of the sea urchin Tripneustes gratilla, reactivated at different concentrations of ATP, were held by suction in the tip of a micropipette and vibrated laterally with respect to the head axis. This imposed vibration resulted in a stable rhythmic beating of the reactivated flagella that was synchronized to the frequency of the micropipette. The reactivated flagella, which in the absence of imposed vibration had an average beat frequency of 39 Hz at 2 mmol l-1 ATP, showed stable beating synchronized to the pipette vibration over a range of 20-70 Hz. Vibration frequencies above 70 Hz caused irregular, asymmetrical beating, while those below 20 Hz induced instability of the beat plane. At ATP concentrations of 10-100 mumol l-1, the range of vibration frequency capable of maintaining stable beating was diminished; an increase in ATP concentration above 2 mmol l-1 had no effect on the range of stable beating. In flagella reactivated at ATP concentrations above 100 mumol l-1, the apparent time-averaged sliding velocity of axonemal microtubules decreased when the imposed frequency was below the undriven flagellar beat frequency, but at higher imposed frequencies it remained constant, with the higher frequency being accompanied by a decrease in bend angle. This maximal sliding velocity at 2 mmol l-1 ATP was close to the sliding velocity in the distal region of live spermatozoa, possibly indicating that it represents an inherent limit in the velocity of active sliding.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Extragenic suppressors of paralyzed flagellar mutations in Chlamydomonas reinhardtii identify loci that alter the inner dynein arms.

1992 ◽  
Vol 118 (5) ◽  
pp. 1163-1176 ◽  
Author(s):  
M E Porter ◽  
J Power ◽  
S K Dutcher
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Synergistic Effects ◽  
Beat Frequency ◽  
Suppressor Mutation ◽  
Swimming Velocity ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Flagellar Beat ◽  
Extragenic Suppressors

We have analyzed extragenic suppressors of paralyzed flagella mutations in Chlamydomonas reinhardtii in an effort to identify new dynein mutations. A temperature-sensitive allele of the PF16 locus was mutagenized and then screened for revertants that could swim at the restrictive temperature (Dutcher et al. 1984. J. Cell Biol. 98:229-236). In backcrosses of one of the revertant strains to wild-type, we recovered both the original pf16 mutation and a second, unlinked suppressor mutation with its own flagellar phenotype. This mutation has been identified by both recombination and complementation tests as a new allele of the previously uncharacterized PF9 locus on linkage group XII/XIII. SDS-PAGE analysis of isolated flagellar axonemes and dynein extracts has demonstrated that the pf9 strains are missing four polypeptides that form the I1 inner arm dynein subunit. The primary effect of the loss of the I1 subunit is a decrease in the forward swimming velocity due to a change in the flagellar waveform. Both the flagellar beat frequency and the axonemal ATPase activity are nearly wild-type. Examination of axonemes by thin section electron microscopy and image averaging methods reveals that a specific domain of the inner arm complex is missing in the pf9 mutant strains (see accompanying paper by Mastronarde et al.). When combined with other flagellar defects, the loss of the I1 subunit has synergistic effects on both flagellar assembly and flagellar motility. These synthetic phenotypes provide a screen for new suppressor mutations in other loci. Using this approach, we have identified the first interactive suppressors of a dynein arm mutation and an unusual bypass suppressor mutation.

Peptide-induced hyperactivation-like vigorous flagellar movement in starfish sperm

Zygote ◽  
2006 ◽  
Vol 14 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Kogiku Shiba ◽  
Tomoko Tagata ◽  
Junko Ohmuro ◽  
Yoshihiro Mogami ◽  
Midori Matsumoto ◽  
...  
Keyword(s):  
Acrosome Reaction ◽  
Sea Urchins ◽  
Beat Frequency ◽  
Swimming Velocity ◽  
Jelly Coat ◽  
Cgmp Signalling ◽  
Flagellar Beat ◽  
Aphelasterias Japonica ◽  
Significant Difference ◽  
Egg Jelly

Asterosap, a sperm-activating peptide (SAP) from the starfish egg jelly coat, is diffusible and controls a cGMP-signalling pathway in starfish sperm in the same manner as resact, a potent chemoattracting SAP in sea urchins. This fact suggests that asterosap may serve as a chemoattractant like resact at concentrations with appropriate gradients. Since asterosap is one of three egg jelly components, which in concert induce the acrosome reaction, it is still worthwhile to evaluate how asterosap modulates sperm motility prior to this reaction. We analysed the flagellar movement of sperm of the starfish Aphelasterias japonica in artificial seawater (ASW) containing the asterosap isoform P15 at 1 μmol l−1. We found that sperm swim straighter with more symmetrical flagellar movement in P15 than in ASW, but without any significant difference in the flagellar beat frequency and the swimming velocity. The flagellar movement is, however, dramatically different between sperm firmly attached to the solid surface by the head in P15 and those attached in ASW: in P15 the flagellum bends to a greater extent, with higher curvature and with higher shear angle up to a right angle to the flagellar wave axis, and beats at an increased frequency. The vigorous flagellar movement of sperm, which can be activated when sperm are placed in high-load circumstances just as entering into a jelly layer, may increase propulsive forces and hydrodynamic resistances, allowing sperm to undergo the acrosome reaction as effectively as possible.

scholarly journals Characterization of a Chlamydomonas Insertional Mutant that Disrupts Flagellar Central Pair Microtubule-associated Structures

1999 ◽  
Vol 144 (2) ◽  
pp. 293-304 ◽  
Author(s):  
David R. Mitchell ◽  
Winfield S. Sale
Keyword(s):  
Beat Frequency ◽  
Live Cells ◽  
Flagellar Motility ◽  
Wild Type ◽  
Electron Microscopic ◽  
Central Pair ◽  
Radial Spoke ◽  
Flagellar Beat ◽  
Sds Page

Two alleles at a new locus, central pair–associated complex 1 (CPC1), were selected in a screen for Chlamydomonas flagellar motility mutations. These mutations disrupt structures associated with central pair microtubules and reduce flagellar beat frequency, but do not prevent changes in flagellar activity associated with either photophobic responses or phototactic accumulation of live cells. Comparison of cpc1 and pf6 axonemes shows that cpc1 affects a row of projections along C1 microtubules distinct from those missing in pf6, and a row of thin fibers that form an arc between the two central pair microtubules. Electron microscopic images of the central pair in axonemes from radial spoke–defective strains reveal previously undescribed central pair structures, including projections extending laterally toward radial spoke heads, and a diagonal link between the C2 microtubule and the cpc1 projection. By SDS-PAGE, cpc1 axonemes show reductions of 350-, 265-, and 79-kD proteins. When extracted from wild-type axonemes, these three proteins cosediment on sucrose gradients with three other central pair proteins (135, 125, and 56 kD) in a 16S complex. Characterization of cpc1 provides new insights into the structure and biochemistry of the central pair apparatus, and into its function as a regulator of dynein-based motility.

Role of the Cilia Membrane in Control of Microtubule Sliding

1980 ◽  
Vol 38 ◽  
pp. 612-615
Author(s):  
Edna S. Kaneshiro
Keyword(s):  
Control Mechanism ◽  
Beat Frequency ◽  
Surface Membrane ◽  
Ciliary Membrane ◽  
Ciliary Beating ◽  
Ciliary Reversal ◽  
Voltage Dependent ◽  
Reversal Mechanism ◽  
And Function

It is currently believed that ciliary beating results from microtubule sliding which is restricted in regions to cause bending. Cilia beat can be modified to bring about changes in beat frequency, cessation of beat and reversal in beat direction. In ciliated protozoans these modifications which determine swimming behavior have been shown to be related to intracellular (intraciliary) Ca2+ concentrations. The Ca2+ levels are in turn governed by the surface ciliary membrane which exhibits increased Ca2+ conductance (permeability) in response to depolarization. Mutants with altered behaviors have been isolated. Pawn mutants fail to exhibit reversal of the effective stroke of ciliary beat and therefore cannot swim backward. They lack the increased inward Ca2+ current in response to depolarizing stimuli. Both normal and pawn Paramecium made leaky to Ca2+ by Triton extrac¬tion of the surface membrane exhibit backward swimming only in reactivating solutions containing greater than IO-6 M Ca2+ Thus in pawns the ciliary reversal mechanism itself is left operational and only the control mechanism at the membrane is affected. The topographic location of voltage-dependent Ca2+ channels has been identified as a component of the ciliary mem¬brane since the inward Ca2+ conductance response is eliminated by deciliation and the return of the response occurs during cilia regeneration. Since the ciliary membrane has been impli¬cated in the control of Ca2+ levels in the cilium and therefore is the site of at least one kind of control of microtubule sliding, we have focused our attention on understanding the structure and function of the membrane.

scholarly journals Development and characterization of a monoclonal antibody blocking human TRPM4 channel

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
See Wee Low ◽  
Yahui Gao ◽  
Shunhui Wei ◽  
Bo Chen ◽  
Bernd Nilius ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Rat Model ◽  
Therapeutic Potential ◽  
Monovalent Cation ◽  
Transgenic Animal ◽  
Cell Swelling ◽  
Live Cells ◽  
Prolonged Incubation ◽  
Trpm4 Channel

AbstractTRPM4 is a calcium-activated non-selective monovalent cation channel implicated in diseases such as stroke. Lack of potent and selective inhibitors remains a major challenge for studying TRPM4. Using a polypeptide from rat TRPM4, we have generated a polyclonal antibody M4P which could alleviate reperfusion injury in a rat model of stroke. Here, we aim to develop a monoclonal antibody that could block human TRPM4 channel. Two mouse monoclonal antibodies M4M and M4M1 were developed to target an extracellular epitope of human TRPM4. Immunohistochemistry and western blot were used to characterize the binding of these antibodies to human TRPM4. Potency of inhibition was compared using electrophysiological methods. We further evaluated the therapeutic potential on a rat model of middle cerebral artery occlusion. Both M4M and M4M1 could bind to human TRPM4 channel on the surface of live cells. Prolonged incubation with TRPM4 blocking antibody internalized surface TRPM4. Comparing to M4M1, M4M is more effective in blocking human TRPM4 channel. In human brain microvascular endothelial cells, M4M successfully inhibited TRPM4 current and ameliorated hypoxia-induced cell swelling. Using wild type rats, neither antibody demonstrated therapeutic potential on stroke. Human TRPM4 channel can be blocked by a monoclonal antibody M4M targeting a key antigenic sequence. For future clinical translation, the antibody needs to be humanized and a transgenic animal carrying human TRPM4 sequence is required for in vivo characterizing its therapeutic potential.

Effect of imposed head vibration on the stability and waveform of flagellar beating in sea urchin spermatozoa

1991 ◽  
Vol 156 (1) ◽  
pp. 63-80 ◽  
Author(s):  
C. Shingyoji ◽  
I. R. Gibbons ◽  
A. Murakami ◽  
K. Takahashi
Keyword(s):  
Sea Urchin ◽  
Beat Frequency ◽  
Distal Region ◽  
Proximal Region ◽  
Bend Angle ◽  
Vibration Frequencies ◽  
Sliding Velocity ◽  
Additional Energy ◽  
The Stability ◽  
Cyclic Changes

The heads of live spermatozoa of the sea urchin Hemicentrotus pulcherrimus were held by suction in the tip of a micropipette mounted on a piezoelectric device and vibrated either laterally or axially with respect to the head axis. Within certain ranges of frequency and amplitude, lateral vibration of the pipette brought about a stable rhythmic beating of the flagella in the plane of vibration, with the beat frequency synchronized to the frequency of vibration [Gibbons et al. (1987), Nature 325, 351–352]. The sperm flagella, with an average natural beat frequency of 48 Hz, showed stable beating synchronized to the pipette vibration over a range of 35–90 Hz when the amplitude of vibration was about 20 microns or greater. Vibration frequencies below this range caused instability of the beat plane, often associated with irregularities in beat frequency. Frequencies above about 90 Hz caused irregular asymmetrical flagellar beating with a marked decrease in amplitude of the propagated bends and a skewing of the flagellar axis towards one side; the flagella often stopped in a cane shape. In flagella that were beating stably under imposed vibration, the wavelength was reduced at higher frequencies and increased at lower frequencies. When the beat frequency was equal to or lower than the natural beat frequency, the apparent time-averaged sliding velocity of axonemal microtubules, obtained as twice the product of frequency and bend angle, decreased with beat frequency in both the proximal and distal regions of the flagella. However, at vibration frequencies above the natural beat frequency, the sliding velocity increased with frequency only in the proximal region of the flagellum and remained essentially unchanged in more distal regions. This apparent limit to the velocity of sliding in the distal region may represent an inherent limit in the intrinsic velocity of active sliding, while the faster sliding observed in the proximal region may be a result of passive sliding or elastic distortion of the microtubules induced by the additional energy supplied by the vibrating pipette. Axial vibration with frequencies either close to or twice the natural beat frequency induced cyclic changes in the waveform, compressing and expanding the bends in the proximal region, but did not affect bends in the distal region or alter the beat frequency.

Function of the Anuran Conus Arteriosus

1974 ◽  
Vol 61 (2) ◽  
pp. 503-520
Author(s):  
R. W. MORRIS
Keyword(s):  
Direct Observation ◽  
Pressure Pulse ◽  
Beat Frequency ◽  
Structure And Function ◽  
Important Measure ◽  
Additional Control ◽  
And Function ◽  
Blood Volumes

1. The anuran conus arteriosus was studied by direct observation of its structure and function and by observing the effects of artificially induced contraction on the pressure pulse in the arterial arches. 2. The beat of the anuran conus was found to close the outlet to the pulmocutaneous arches. The timing of the beat of the conus in the ventricular cycle is variable; early in the cycle the conus admits less blood to the pulmocutaneous circuit, more blood being admitted when it beats late. 3. The timing of the beat of the conus appears to be adaptively related to beat frequency and to pH of the system. Elementary calculations suggest that these characteristics constitute an important measure of survival value of the anuran conus. 4. The findings of this work are compatible with the theory of selective passage of two streams through the heart, supplementing such passage with additional control of blood volumes entering the pulmocutaneous circuit.

Polyglutamylation and polyglycylation of alpha- and beta-tubulins during in vitro ciliated cell differentiation of human respiratory epithelial cells

1999 ◽  
Vol 112 (23) ◽  
pp. 4357-4366 ◽  
Author(s):  
K. Million ◽  
J. Larcher ◽  
J. Laoukili ◽  
D. Bourguignon ◽  
F. Marano ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Differentiation ◽  
Epithelial Cells ◽  
Beat Frequency ◽  
Ciliated Cell ◽  
Early Event ◽  
Functional Assay ◽  
Ciliated Cells ◽  
Human Nasal Epithelial Cells ◽  
Centriole Assembly

Tubulins are the major proteins within centriolar and axonemal structures. In all cell types studied so far, numerous alpha- and beta-tubulin isoforms are generated both by expression of a multigenic family and various post-translational modifications. We have developed a primary culture of human nasal epithelial cells where the ciliated cell differentiation process has been observed and quantified. We have used this system to study several properties concerning polyglutamylation and polyglycylation of tubulin. GT335, a monoclonal antibody directed against glutamylated tubulins, stained the centriole/basal bodies and the axonemes of ciliated cells, and the centrioles of non-ciliated cells. By contrast, axonemal but not centriolar tubulins were polyglycylated. Several polyglutamylated and polyglycylated tubulin isotypes were detected by two-dimensional electrophoresis, using GT335 and a specific monoclonal antibody (TAP952) directed against short polyglycyl chains. Immunoelectron microscopy experiments revealed that polyglycylation only affected axonemal tubulin. Using the same technical approach, polyglutamylation was shown to be an early event in the centriole assembly process, as gold particles were detected in fibrogranular material corresponding to the first cytoplasmic structures involved in centriologenesis. In a functional assay, GT335 and TAP952 had a dose-dependent inhibitory effect on ciliary beat frequency. TAP952 had only a weak effect while GT335 treatment led to a total arrest of beating. These results strongly suggest that in human ciliated epithelial cells, tubulin polyglycylation has only a structural role in cilia axonemes, while polyglutamylation may have a function both in centriole assembly and in cilia activity.

Temperature effects on anaphase chromosome movement in the spermatocytes of two species of crane flies (Nephrotoma suturalis Loew and Nephrotoma ferruginea Fabricius)

1979 ◽  
Vol 39 (1) ◽  
pp. 29-52
Author(s):  
C.J. Schaap ◽  
A. Forer
Keyword(s):  
Sex Chromosomes ◽  
Temperature Effects ◽  
Sex Chromosome ◽  
Beat Frequency ◽  
General Pattern ◽  
Force Production ◽  
Temperature Shift ◽  
Ciliary Beat Frequency ◽  
Chromosome Movement ◽  
Flagellar Beat

Using phase-contrast cinemicrography on living crane fly (Nephrotoma suturalis Loew and Nephrotoma ferruginea Fabricius) spermatocytes, we have studied the effects of a range of temperatures (6–30 degrees C) on the anaphase I chromosome-to-pole movements of both autosomes and sex chromosomes. In contrast to previous work we have been able to study chromosome-to-pole velocities of autosomes without concurrent pole-to-pole elongation. In these cells we found that the higher the temperature, the faster was the autosomal chromosomes movement. From reviewing the literature we find that the general pattern of the effects of temperature on chromosome movement is similar whether or not pole-to-pole elongation occurs simultaneously with the chromosome-to-pole movement. Changes in cellular viscosities calculated from measurements of particulate Brownian movement do not seem to be able to account for the observed velocity differences due to temperature. Temperature effects on muscle contraction speed, flagellar beat frequency, ciliary beat frequency, granule flow in nerves, and chromosome movement have been compared, as have the activation energies for the rate-limiting steps in these motile systems: no distinction between possible mechanisms of force production is possible using these comparisons. The data show that even the different autosomes within single spermatocytes usually move at different speeds. These velocity differences cannot simply be related to chromosome size as the autosomes are visually indistinguishable. The sex chromosomes start their anaphase poleward movement after that of the autosomes, and move more slowly (by a factor of about 4), but their velocities appear to be affected by temperature in the same fashion as those of the autosomes. The interval between the onset of autosome anaphase and sex chromosome anaphase is also affected by temperature: the higher the temperature, the shorter the interval between the 2 stages. We have observed abnormalities in sex chromosome segregation, which may be due to temperature, but have not determined what the exact temperature shift conditions are that cause these abnormalities.

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