mouse lens dissection and processing to study complex cell-to-cell interdigitations and the membrane architecture

Immunofluorescence Staining of Membrane Interdigitations in Lens Fiber Cells

The lens is a clear and ovoid tissue in the anterior chamber of the eye that is made up of two cell types, epithelial and fiber cells1 (Figure 1). There is a monolayer of epithelial cells that covers the anterior hemisphere of the lens. Fiber cells are differentiated from epithelial cells and make up the bulk of the lens. The highly specialized fiber cells undergo an elongation, differentiation, and maturation programming, marked by distinct changes in cell membrane morphology from the lens periphery to the lens center2,3,4,5,6,7,8,9,10,11,12, also known as the lens nucleus. The function of the lens to fine-focus light coming from various distances onto the retina depends on its biomechanical properties, including stiffness and elasticity13,14,15,16,17,18,19. The complex interdigitations of lens fibers have been hypothesized20,21 and recently shown to be important for lens stiffness22,23.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65638/65638fig01.jpg" /> 
Figure 1: Lens anatomy diagrams and representative scanning electron microscopy (SEM) images from lens fibers. The cartoon shows a longitudinal (anterior to posterior from top to bottom) view of the anterior monolayer of epithelial cells (shaded in light blue) and a bulk mass of lens fiber cells (white). The center of the lens (shaded in pink) is known as the nucleus and comprises highly compacted fiber cells. On the right, a cross-section cartoon reveals the elongated hexagon cell shape of lens fibers that are packed into a honeycomb pattern. Fiber cells have two broad sides and four short sides. Representative SEM images along the bottom show the complex membrane interdigitations between lens fiber cells at different depths of the lens. From the right, newly formed lens fibers at the lens periphery have small protrusions along the short sides and balls-and-sockets along the broad side (red boxes). During maturation, lens fibers develop large paddle domains that are decorated by small protrusions along the short sides (blue boxes). Mature fiber cells possess large paddle domains illustrated by small protrusions. These interlocking domains are important for lens biomechanical properties. Fiber cells in the lens nucleus have fewer small protrusions along their short sides and have complex tongue-and-groove interdigitations (purple boxes). The broad sides of the cell display a globular membrane morphology. The cartoon was modified from22,32 and not drawn to scale. Scale bar = 3 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65638/65638fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The lens grows by adding shells of new fiber cells overlaid on top of previous generations of fibers24,25. Fiber cells have an elongated, hexagonal cross section shape with two broad sides and four short sides. These cells extend from the anterior to the posterior pole of the lens, and depending on the species, the lens fibers can be several millimeters in length. To support the structure of these elongated and skinny cells, specialized interdigitations along the broad and short sides create interlocking structures to maintain the lens shape and biomechanical properties. Changes in cell membrane shape during fiber cell differentiation and maturation have been extensively documented by electron microscopy (EM) studies2,3,4,5,6,7,8,9,10,20,26,27,28,29. Newly formed fiber cells have balls-and-sockets along their broad sides with very small protrusions along their short sides, while mature fibers have interlocking protrusions and paddles along their short sides. Nuclear fibers display tongue-and-groove interdigitations and globular membrane morphology. Little is known about the proteins that are required for these complex interlocking membranes. Previous studies on protein localization in fiber cells have relied on lens tissue sections, which do not allow clear visualization of the complex cell architecture.
This work has created and perfected a novel method to fix single and bundles of lens fiber cells to preserve the complex morphology and to allow immunostaining for proteins at the cell membrane and within the cytoplasm. This method faithfully preserves cell membrane architecture, comparable to data from EM studies, and allows staining with primary antibodies for specific proteins. We have previously immunostained cortical lens fibers undergoing differentiation and maturation22,23. In this protocol, there is also a new method to stain fiber cells from the lens nucleus. This protocol opens the door to understanding the mechanisms for formation and changes in membrane interdigitations during fiber cell maturation and lens nucleus compaction.

Author Spotlight: Advancing Lens Biomechanics Research Through a Novel Protocol for Imaging Complex Interdigitations and Protein Staining 

Preparation and Immunofluorescence Staining of Bundles and Single Fiber Cells from the Cortex and Nucleus of the Eye Lens

The lens is a transparent ellipsoid organ in the anterior chamber of the eye, and is responsible for fine focusing of light onto the retina to create a clear image. The function of the lens relies on its biomechanical properties, complex interdigitations between the lens fiber cells has recently been shown to be important for lens stiffness. While the specialized morphology of lens fiber cells has been previously described by electron microscopy, little is known about the proteins that are required for these interlocking membranes.Previous studies have relied on lens tissue sections, which do not allow clear visualization of complex cell architecture. We have created and perfected a novel technique to faithfully preserve the complex membrane interdigitations between lens fiber cells for staining and confocal microscopy imaging of specific proteins. In this protocol, there is also a new method for staining the fiber cells from the center of the lens, which is also known as the lens nucleus.This method opens the door to understanding fiber cell differentiation and maturation by studying the mechanisms of formation and changes in membrane interdigitations.

Watch this Scientific Journal Video about Mouse Lens Dissection and Processing to Study Complex Cell-to-Cell Interdigitations and the Membrane Architecture at JoVE.com

Mouse Lens Dissection and Processing to Study Complex Cell-to-Cell Interdigitations and the Membrane Architecture

Begin by positioning a euthanized mouse for eye enucleation using curved forceps to press the tissue around the eye to displace the eye out of the socket. Then lift and remove the eye, and transfer it to fresh PBS in the dissection tray. Using ultra fine scissors, cut the optic nerve as close as possible to the eyeball, and carefully insert fine tip straight tweezers into the eyeball through the optic nerve's exit at the posterior of the eye.Now, insert scissors at the posterior of the eye and begin making an incision from the posterior toward the corneal scleral junction, and continue the incision until half of the junction has been separated. Gently push on the cornea so that the lens can exit through the incision. Using fine tip straight tweezers, carefully remove any large pieces of tissue from the lens.After finding the equatorial region, shallowly pierce the lens, and then remove the lens capsule. Transfer the lens fiber cells to a 60 millimeter dish with 1%paraformaldehyde. Using a sharp scalpel, split the ball of the fiber cells in half along its anterior posterior axis.And then further cut the halves along the same axis to produce quarters. Use the straight tweezers to remove the nucleus region from the lens fiber cell quarter. Next, add 200 microliters of 1%paraformaldehyde to a 48-well plate.Transfer the lens cortex to the plate and incubate for 15 minutes at room temperature with gentle shaking at 300 RPM. After blocking, add appropriate primary antibodies to the 48-well plate and transfer samples to the primary antibody solution. Incubate the samples overnight at 4 degrees Celsius with gentle shaking or mutation.The next day, wash the samples with PTX, and similarly, incubate them with secondary antibodies. After washing the samples, add one drop or 50 microliters of mounting media onto a plus charged microscope slide. Place the tissue in the mounting media.And use tweezers to gently separate the fiber cells from each other. Then, gently place a cover slip on top of the separated cells and mounting media. After removing excess media, use nail polish to seal the edges of the cover slip on the slide before confocal microscopy.After the lens dissection and lens capsule removal, transfer the fiber cell mass to wet gloved fingertips and gently roll in all directions to separate the lens nucleus. Then, transfer the lens nucleus to freshly made 1%paraformaldehyde solution in a 48-well plate and incubate overnight at 4 degrees Celsius with gentle shaking or mutation. The next day, transfer the sample to a 60 millimeter dish with 1%paraformaldehyde and use a sharp scalpel to split the lens nucleus along the anterior posterior axis in half, then into quarters.Then, postfix, block, stain, and mount the sample as previously demonstrated In these preparations, bundles of lens fiber cells with unique morphologies are found from different lens regions. The staining of the F-actin network shows enrichment at the cell membrane in differentiating and mature fibers while F-actin signals are present in the cytoplasm of nuclear fibers. Scanning electron microscopy and confocal images of differentiating fiber cells show ball and socket interdigitations with small interlocking protrusions, whereas mature fibers have paddles decorated by small protrusions.Scanning electron microscopy and confocal microscope images of nuclear lens fiber cells reveal infrequent and larger interlocking protrusions on the short sides of the cells where the cell membrane is rough and has tongue and groove interdigitations in these cells from the center of the lens.

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