Dennis E. Koppel

A soluble protein is immobile in dormant spores of Bacillus subtilis but is mobile in germinated spores: Implications for spore dormancy

Fluorescence redistribution after photobleaching has been used to show that a cytoplasmic GFP fusion is immobile in dormant spores of Bacillus subtilis but becomes freely mobile in germinated spores in which cytoplasmic water content has increased ≈2-fold. The GFP immobility in dormant spores is not due to the high levels of dipicolinic acid in the spore cytoplasm, because GFP was also immobile in germinated cwlD spores that had excreted their dipicolinic acid but where cytoplasmic water content had only increased to a level similar to that in dormant spores of several other Bacillus species. The immobility of a normally mobile protein in dormant wild-type spores and germinated cwlD spores is consistent with the lack of metabolism and enzymatic activity in these spores and suggests that protein immobility, presumably due to low water content, is a major reason for the metabolic dormancy of spores of Bacillus species.

Diffusion of dihydropyridine calcium channel antagonists in cardiac sarcolemmal lipid multibilayers.

A membrane bilayer pathway model has been proposed for the interaction of dihydropyridine (DHP) calcium channel antagonists with receptors in cardiac sarcolemma (Rhodes, D.G., J.G. Sarmiento, and L.G. Herbette. 1985. Mol. Pharmacol. 27:612-623) involving drug partition into the bilayer with subsequent receptor binding mediated (though probably not rate-limited) by diffusion within the bilayer. Recently, we have characterized the partition step, demonstrating that DHPs reside, on a time-average basis, near the bilayer hydrocarbon core/water interface. Drug distribution about this interface may define a plane of local concentration for lateral diffusion within the membrane. The studies presented herein examine the diffusional dynamics of an active rhodamine-labeled DHP and a fluorescent phospholipid analogue (DiIC16) in pure cardiac sarcolemmal lipid multibilayer preparations as a function of bilayer hydration. At maximal bilayer hydration, the drug diffuses over macroscopic distances within the bilayer at a rate identical to that of DiI (D = 3.8 X 10(-8) cm2/s), demonstrating the overall feasibility of the membrane diffusion model. The diffusion coefficients for both drug and lipid decreased substantially as the bilayers were dehydrated. While identical at maximal hydration, drug diffusion was significantly slower than that of DiIC16 in partially dehydrated bilayers, probably reflecting differences in mass distribution of these probes in the bilayer.

Migration of the guinea pig sperm membrane protein PH-20 from one localized surface domain to another does not occur by a simple diffusion-trapping mechanism

The redistribution of membrane proteins on the surface of cells is a prevalent feature of differentiation in a variety of cells. In most cases the mechanism responsible for such redistribution is poorly understood. Two potential mechanisms for the redistribution of surface proteins are: (1) passive diffusion coupled with trapping, and (2) active translocation. We have studied the process of membrane protein redistribution for the PH-20 protein of guinea pig sperm, a surface protein required for sperm binding to the egg zona pellucida (P. Primakoff, H. Hyatt, and D. G. Myles (1985). J. Cell Biol. 101, 2239-2244). PH-20 protein is localized to the posterior head plasma menbrane of the mature sperm cell. Following the exocytotic acrosome reaction, PH-20 protein moves into the newly incorporated inner acrosomal membrane (IAM), placing it in a position favorable for a role in binding sperm to the egg zona pellucida (D. G. Myles, and P. Primakoff (1984), J. Cell Biol. 99, 1634-1641). To analyze the mechanistic basis for this protein migration, we have used fluorescence microscopy and digital image processing to characterize PH-20 protein migration in individual cells. PH-20 protein was observed to move against a concentration gradient in the posterior head plasma membrane. This result argues strongly against a model of passive diffusion followed by trapping in the IAM, and instead suggests that an active process serves to concentrate PH-20 protein toward the boundary separating the posterior head and IAM regions. A transient gradient of PH-20 concentration observed in the IAM suggests that once PH-20 protein reaches the IAM, it is freely diffusing. Additionally, we observed that migration of PH-20 protein was calcium dependent.

Rearrangement of Sperm Surface Antigens prior to Fertilization

During spermiogenesis and epididymal transit, proteins on the sperm surface become localized to specific domains. In at least one case (PH-20), the protein is initially inserted throughout the membrane and subsequently becomes restricted to a domain by some mechanism that has not yet been determined. Other proteins could become localized through localized insertion. The sperm surface is a dynamic structure that is altered even after the spermatozoon leaves the male. In the female reproductive tract the spermatozoa undergo capacitation and the acrosome reaction that enables them to fertilize the egg. Both of these processes are accompanied by alterations in protein localization: the PT-1 protein migrates during capacitation, and the PH-20 protein migrates after the acrosome reaction. In addition, an upregulation of the surface expression of PH-20 occurs during the acrosome reaction. This additional PH-20 is incorporated into the plasma membrane by the irreversible fusion of the acrosomal membrane with the plasma membrane. The acrosomal membrane contains PH-20 protein that has been stored there since the formation of the acrosome at the spermatid stage of spermiogenesis. Proteins that are freely diffusing must be maintained in a domain by a mechanism that does not involve immobilization or slowing of protein diffusion. We have suggested that barriers to membrane protein diffusion exist at the equatorial region, the posterior ring, and the annulus and that they are responsible for maintaining a localized distribution of at least some of the surface proteins. The migration of surface proteins could result from an alteration of these barriers, a change in the protein structure so that it can pass through the barrier, or active transport across the barrier. These observed changes in surface expression (localization and the level of expression) may be acting to control surface function post-testicularly.

Barriers to diffusion of plasma membrane proteins form early during guinea pig spermiogenesis.

The plasma membrane of the mature guinea pig sperm is segregated into at least four domains of different composition. Previous studies have shown that some proteins localized within these domains are free to diffuse laterally, suggesting that barriers to protein diffusion are responsible for maintaining the nonuniform distribution of at least some surface proteins in mature sperm. The different membrane domains appear sequentially during sperm morphogenesis in the testis and during later passage through the epididymis. To determine when diffusion barriers become functional during sperm development, we examined the diffusion of two proteins that are expressed on the cell surface of developing spermatids and become segregated to different plasma membrane domains during the course of spermiogenesis. Both proteins exhibited rapid lateral diffusion throughout spermiogenesis, even after they become localized to specific regions of the surface membrane. These results suggest that barriers to membrane diffusion form concomitantly with membrane domains during spermiogenesis.

Analysis of heterogeneous fluorescence photobleaching by video kinetics imaging: The method of cumulants

The method of cumulants has been applied to digital video fluorescence microscopy. The method is used to reconstruct the distribution of fluorescent molecules before the initiation of fluorescence photobleaching, and to characterize heterogeneous photobleaching by imaging one or more of the cumulants of the bleaching decay rate. Using the pipelined pixel processor of the image analysis system for the bulk of the calculations, rather than the general‐purpose host‐computer CPU, the video kinetics imaging can be performed in near real‐time. The method is applied to chick embryo myotubes labelled with fluorescein‐conjugated α‐bungarotoxin. The pre‐bleach fluorescence distribution is derived, and the image of fluorescein fluorescence is separated from glutaraldehyde‐induced autofluorescence on the basis of the spatially resolved average photobleaching decay rate.

Cyclic 3′,5′-AMP Causes ADAM1/ADAM2 to Rapidly Diffuse Within the Plasma Membrane of Guinea Pig Sperm

Abstract Because sperm cannot synthesize new proteins as they journey to the egg, they use multiple mechanisms to modify the activity of existing proteins, including changes in the diffusion coefficient of some membrane proteins. Previously, we showed that during capacitation the guinea pig heterodimeric membrane protein ADAM1/ADAM2 (fertilin) transforms from a stationary state to one of rapid diffusion within the lipid bilayer. The cause for this biophysical change, however, was unknown. In this study we examined whether an increase in cAMP, such as occurs during capacitation, could trigger this change. We incubated guinea pig cauda sperm with the membrane-permeable cAMP analog dibutyryl cAMP (db-cAMP) and the phosphodiesterase inhibitor papaverine and first tested for indications of capacitation. We observed hypermotility and acrosome-reaction competence. We then used fluorescence redistribution after photobleaching (FRAP) to measure the lateral mobility of ADAM1/ADAM2 after the db-cAMP treatment. We observed that db-cAMP caused roughly a 12-fold increase in lateral mobility of ADAM1/ADAM2, yielding diffusion similar to that observed for sperm capacitated in vitro. When we repeated the FRAP on testicular sperm incubated in db-cAMP, we found only a modest increase in lateral mobility of ADAM1/ADAM2, which underwent little redistribution. Interestingly, testicular sperm also cannot be induced to undergo capacitation. Together, the data suggest that the release of ADAM1/ADAM2 from its diffusion constraints results from a cAMP-induced signaling pathway that, like others of capacitation, is established during epididymal sperm maturation..

Fluorescence Techniques for the Study of Biological Motion

A variety of fluorescence techniques for the study of biological motion are described and discussed. We consider three basic strategies: time-resolved emission, fluorescence photobleaching, and fluorescence correlation spectroscopy. In the first of these, the sample is excited with pulses of light short compared to the excited singlet-state lifetime. Molecular motions over the time-scale of the excited state lifetime are characterized through their effects on the observed time-resolved fluorescence emission. In the fluorescence photobleaching technique, intense photobleaching pulses, short compared to the time-scale of the motion under study (but very long compared to the excited singlet-state lifetime), are used to selectively deplete the ground-state population. Molecular motions over times long compared to the excited singlet-state lifetime are characterized by measuring the “recovery” of fluorescence after photobleaching monitored with an attenuated CW light source. In fluorescence correlation spectroscopy, in contrast, the sample is illuminated only with the CW monitoring beam. Molecular motions over times long compared to the excited-state lifetime are characterized through calculations of the correlation function of the spontaneous, stochastic fluorescence intensity fluctuations about the ensemble average.