Filipin Labeling Research Articles

Filipin-sterol complexes at nodes of Ranvier.

Using the filipin-sterol technique, regional heterogeneity in the axonal and Schwann cell plasma membranes was investigated at the node of Ranvier and paranodes. Filipin-sterol complexes were abundant at the nodal axolemma but infrequent throughout the paranodal axolemma. The paranodal Schwann cell plasma membrane was rich in complexes which extended over the nodal Schwann cell microvilli. There were no regional differences in filipin labelling of the nodal-paranodal Schwann cell plasma membrane in relation to features such as paranodal cytoplasmic columns or mesaxonal furrows. However, the paranodes of adjacent Schwann cells were sometimes markedly different from each other in the amount of filipin labelling. The extent to which filipin labelling is indicative of cholesterol membrane content is discussed and the findings are related to current concepts of distribution, mobility and interaction of protein and lipid in biomembranes, with particular reference to the nodal axolemma.

Different patterns of filipin-sterol labeling in prostate nuclear membranes from normal and castrated rats

Filipin was used as cytochemical probe for sterol detection in freeze-fractured prostate nuclear membranes from rats under different hormonal conditions. Isolated prostate acini and nuclei were fixed in glutaraldehyde and post-treated with filipin, according to Robinson and Karnovsky (1980). In general, most plasma and intracellular cytoplasmic membranes displayed a marked response to filipin in either epithelial and stromal cells from normal and castrated animals. Nuclear membranes from epithelial secretory cells were systematically negative to filipin labeling in normal animals, although after castration a positive response was detected. Stromal nuclear membranes were labeled both in normal and castrated animals. Filipin-treated isolated nuclei displayed the same overall labeling pattern but there was a different distribution of induced deformations relative to intact cell nuclei. These observations indicate that: a) nuclear membranes from different cell types have different responses to filipin; b) a change in the molecular organization of nuclear membranes from prostate secretory cells follow castration; c) nuclei isolation affects the distribution of filipin induced deformations on the membranes.

Compactin (ML-236B) reduces the content of filipin-cholesterol complexes in the plasma membrane of chronic lymphocytic leukemia cells

The polyene antibiotic filipin was used to visualize the presence and distribution of cholesterol in the plasma membrane of glutaraldehyde-fixed human chronic lymphocytic leukemia (CLL) cells. Both compactin (ML-236B), a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and 25-hydroxycholesterol reduced the content of filipin-cholesterol complexes in the plasma membrane of CLL cells grown in media supplemented with either 15 % delipidized horse serum or 15 % normal (whole) horse serum. The reduction due to compactin was reversed by the concomitant addition of mevalonolactone. The ability of compactin to reduce the relative cholesterol content (as judged by filipin labeling) in CLL cells grown in lipoprotein-containing (normal) serum suggest that either CLL cells are different from other cells in that they predominantly utilize endogenously synthesized cholesterol for incorporation into the plasma membrane, or that a separate pool of endogenously synthesized cholesterol provides cholesterol for the plasma membrane.

Differential filipin labeling of the luminal membranes lining the pancreatic acinus.

The cytochemical labeling of cholesterol by filipin in plasma membranes of the exocrine pancreas has shown a difference in the reactivity to filipin between the acinar and centro-acinar cells. The acinar cell has a poorly labeled luminal membrane but numerous filipin-cholesterol complexes on the basolateral membrane. In contrast, the centro-acinar cell shows a comparable degree of filipin labeling on both luminal and basolateral membrane. This difference in the distribution of filipin labeling suggests underlying differences in the organization of the respective membranes, probably related to their specific functions.

Development of membrane differentiations in the guinea pig spermatid during spermiogenesis.

AbstractWe have studied the development of membrane differentiations in guinea pig spermatids during spermiogenesis, using electron microscopy of thin sections, freeze‐fracturing, and filipin labeling as an indicator of membrane cholesterol distribution. The development of the distinctive membrane specializations closely correlated with the developmental steps of spermatid differentiation. The annulus, the zipper, and the circumferential strands of particles overlying the mitochondrial gyres of the midpiece appeared in the plasma membrane over the tail during the last two steps of spermiogenesis. The outer acrosomal membrane showed trnsient crenations during the acrosome phase and serrations over the equatorial segment of the acrosome during the maturation phase. On the inner nuclear membrane, a depression appeared opposite the inner zone of the acrosome during the Golgi phase and persisted throughout spermiogenesis. The implantation fossa also appeared during this phase, as a bulging on the inner nuclear envelope covered with small membrane particles. By the cap phase, small and large 15–20‐nm particles were present at the implantation‐fossa site, and clusters of 9‐nm particles were seen over the postacrosomal segment of the nuclear envelope. The migration of the nuclear pores began when close contacts were established between adjacent nuclear membranes. It started simultaneously at the anterior pole and the implantation fossa and later progressed over the segment of membrane between the caudal margin of the acrosome and the posterior ring. By the maturation phase, the nuclear pores had migrated to the redundant nuclear envelope.After filipin labeling, a gradient was visible in the distribution of the cholesterol‐filipin complexes, decreasing from the plasma membrane to the nuclear membrane. Within the same membrane, cholesterol distribution varied from one pole of the cell to the other. On the differentiations of the plasma membrane over the tail, cholesterol‐filipin complexes were absent, but they were profuse in the membrane over the head. In the acrosomal membrane, the complexes were gathered in clumps associated with the crenations. They became fewer in the rostral segment but remained moderate in the caudal equatorial segment as spermiogenesis proceeded. The nuclear membrane contained cholesterol‐free regions opposite the inner acrosome and at the site of the implantation fossa. Otherwise, the acrosomal segment of the nuclear envelope demonstrated a homogeneous distribution of cholesterol‐filipin complexes, while the postacrosomal segment showed small clumps of complexes adjacent to nuclear pores. We propose that each of the spermatid cell membranes is not an autonomous system, but is dependent upon its exchanges with the immediate external environment under the one side of the membrane and its close coupling to the internal environment (i.e., membrane‐associated structures: annulus, zipper, etc.) under the otehr side of the membrane. We suggest that the cholesterol in spermatid cell membranes contributes to the establishment of their mosaicism and regulates the modeling of the spermatid by modulating the internal fluidity of individual membrane segments during spermiogenesis.

Filipin labelling of lipid droplets in lactating rat mammary gland

Filipin labelling of lipid droplets in lactating rat mammary gland

Distribution of cytochemically detectable cholesterol in the electric organ of Torpedo marmorata.

A cytochemical probe for cholesterol, the polyene antibiotic filipin, was applied to aldehyde-fixed samples of the electric organ of Torpedo marmorata to identify filipin-binding sites in the various membrane components of the organ and, hence, the probable cholesterol content at these levels. In both thin-sectioned and freeze-fractured samples, filipin-cholesterol complexes appeared numerous and homogeneously distributed on the Schwann cell plasma membrane. On the presynaptic membrane, filipin-cholesterol complexes occurred in patches alternating with unlabeled membrane segments. The postsynaptic, acetylcholine receptor-rich plasma membrane of the electroplax showed no or few filipin-cholesterol complexes in the flat region and upper part of the invaginations (both areas characterized by a lattice of small intramembrane particles); however, the membrane of the bottom part of the postsynaptic invaginations contained several complexes. The ventral, noninnervated plasma membrane of the electroplax showed a moderate, homogeneous filipin labeling. These data suggest that the distribution of cholesterol among membranes of the electroplax is not homogeneous and that the acetylcholine receptor-rich region of the postsynaptic membrane (as characterized by the lattice of small intramembrane particles) may contain little cholesterol.

Freeze-fracture cytochemistry of neuronal membranes: inhomogeneous distribution of filipin-sterol complexes in perikarya, dendrites and axons

Vibratome slices of cerebellar cortex fixed in a glutaraldehyde solution containing filipin, a sterol-specific probe, were freeze-fractured to study the distribution of filipin-sterol complexes in neuronal plasma membranes. These complexes appear as 25–30 nm protuberances or pits in the fracture face of plasma membranes, and their density was low in dendrites of Purkinje and granule cells. In contrast, the plasma membranes of neuronal perikarya showed an abundant filipin labeling, 4–6 times greater than in dendrites. Parallel fibers, the axons of granule cells also had significantly more filipin-sterol complexes than granule cell dendrites, but fewer than granule cell perikarya. These results reveal differences in the organization of specific regions of neuronal plasma membranes, which are also characterized by a different pattern of synaptic contacts.

Topology of morphologically detectable protein and cholesterol in membranes of polypeptide-secreting cells.

The freeze-fracture morphology of intracellular and plasma membranes in endocrine and exocrine polypeptide-secreting cells has been studied to detect changes while these membranes interact during secretion. A qualitative and quantitative evaluation of intramembrane particles and filipin binding as indicators of protein and cholesterol content of the membranes, respectively, reveals the following changes. From the forming of the maturing pole of the Golgi complex, membranes lose morphologically detectable protein and gain morphologically detectable cholesterol. The protein-poor, cholesterol-rich secretory granule membrane then interacts with a richly particulate plasma membrane in endocrine cells and with a moderately particulate luminal membrane in exocrine cells. The site of interaction between secretory granule and plasma membrane is characterized by a local clearing of intramembrane particles; by contrast, filipin-binding sites revealing cholesterol are present in this area. In exocrine cells, the fused secretory granule, which is initially rich in filipin-cholesterol complexes and poor in particles, appears to lose progressively its filipin labelling to resemble the poorly labelled luminal membrane. These findings, although they cannot be interpreted definitely at present, clearly show impressive changes of membrane structure along the secretory pathway and suggest that a corresponding degree of functional specialization is needed for proper interaction to occur.