Mary S. Tilney

Actin filaments, stereocilia, and hair cells of the bird cochlea. III. The development and differentiation of hair cells and stereocilia.

The cochleae of chick embryos of 8 days of incubation until hatching (21 days) were examined by scanning electron microscopy. Unlike what one would expect from the literature, the total number of hair cells per cochlea (10,405 +/- 529) is already determined and visible in a 10-day embryo and the growth of the cochlea is a result of the growth in size and surface area of the hair cells. We also find that the hair cells differentiate simultaneously throughout the cochlea and have followed the differentiation of individual hair cells throughout development. During development we find that the total number, hexagonal packing, and orientation of the stereocilia in each hair cell is determined early and accurately (9- to 10-day embryos). The stereocilia then begin to elongate in all the cells of the cochlea at approximately 0.5 micron/day. By Day 12 the tallest stereocilia in each cell are 1.5-1.8 micron long, the mature length for cells at the proximal end of the cochlea. At this point all stereocilia cease elongating, but those along the inferior edge gradually increase in width from 0.11 micron to maximally 0.19 micron in 17-day embryos. When the stereocilia on the inferior edge reach their mature width, widening ceases and the elongation of stereocilia in the distal hair cells begins again. When these stereocilia have attained their mature lengths, they stop growing. Thus elongation and widening of stereocilia are separated in time. During this period, 11 to 13 days, the shape of the tufts at the proximal end of the cochlea changes. This occurs because stereocilia in the front of each tuft are absorbed while others at the sides appear de novo. This rearrangement converts a circular bundle of stereocilia to a rectangular bundle.

The wily ways of a parasite: induction of actin assembly by Listeria

The intracellular pathogen Listeria has a spectacular mode of transport within and between host cells. By inducing host cell actin to assemble from its surface, the bacterium forms a tail composed of many short, crossbridged actin filaments. With this tail Listeria is propelled across the cytoplasm like a comet streaking across the sky. Here we discuss the antics of Listeria and some of the bacterial genes instrumental in maintaining it in the host.

The distribution of hair cell bundle lengths and orientations suggests an unexpected pattern of hair cell stimulation in the chick cochlea

A detailed analysis of the morphological polarity of the hair cell bundles on the chick cochlea was carried out. Although the pattern is identical from cochlea to cochlea, the morphological polarity of the bundles varies at different positions on the cochlea. More specifically, the hair cell bundles located immediately adjacent to the inferior and superior edges are oriented with their morphological polarity perpendicular to the margins. As we move across the cochlea (transect it), there is a gradual rotation in the polarity of the bundles so that in the center of the cochlea the hair cells are oriented at an angle to those at the edges. As we continue to the superior edge the polarity gradually rotates back again. The amount of rotation depends on the position of the transect such that at the extreme proximal end there is little rotation, while at the distal end the rotation is up to 90 degrees. The rotation is always in the same direction with the tallest rows of stereocilia nearest the distal end of the cochlea. Measurements of the length of the longest stereocilia in the hair cell bundles revealed that not only are the bundles systematically longer from the proximal to distal end of the cochlea, but also the hair cells on the superior edge are significantly longer than those on the inferior edge at the same distance from one end of the cochlea. If we draw on micrographs of the cochlea contour lines through hair cells whose stereocilia are the same height, these lines coincide with the morphological polarity of the hair cells included in these contours. Furthermore analysis of damage to the cochlea induced by pure tones of high intensity also roughly follows the same contour lines. We conclude that unlike what has been thought, the stimulation of hair cells by pure tones may not occur in a strictly transverse pattern, but instead may follow the oblique contours demonstrated here.

Functional organization of the cytoskeleton

Within each stereocilium of chick hair cells is a hexagonally packed bundle of actin filaments. Diffraction patterns of thin sections of these bundles reveal that the actin filaments are aligned such that the crossover points of adjacent filaments are in transverse register. Since each actin filament is composed of subunits that are organized in a helical pattern, yet all the actin filaments are in transverse register, crossbridges between filaments can form only at positions dictated by the geometry of the actin helix or at 125 A intervals. Thus the crossbridges appear in electron micrographs as regularly spaced bands (125 A) that are perpendicular to the axis of the stereocilium. From examination of stereocilia of organisms who have a temporary threshold shift due to exposure to loud noise, we know that the integrity of the actin filaments and their crossbridges is essential for hair cell function. However, particularly interesting is that when a stereocilium is bent or displaced, as might occur during stimulation by sound, the actin filaments are not compressed or stretched, but slide past one another so that the bridges become tilted relative to the long axis of the actin filament bundle. Thus, resistance to bending or displacement must be a property of the number of bridges present which in turn is a function of the number and lengths of actin filaments present. Since hair cells in different parts of the cochlea have stereocilia of different, yet predictable lengths and widths, this means that the force needed to displace the stereocilia of hair cells located at different regions of the cochlea will not be the same. This suggests that fine tuning of the hair cells must be a built-in property of the stereocilia. To try to understand how hair cells control the length and number of actin filaments per stereocilium and thus the length and width of the stereocilia, we examined cochlea in chick embryos of increasing maturity. Of interest is that very early in development (10-day embryos) the total hair cell number and position is specified. Thus it is possible to study the growth of stereocilia in cells whose final stereociliary length and width is already known. Stereocilia first elongate (from 8 to 11 days--first phase); they then stop elongating and increase in width (12-16 days--second phase), then elongate again (third phase) to the length appropriate to the position of the hair cell on the cochlea. During the first phase a few actin filaments are present, but initially poorly ordered.(ABSTRACT TRUNCATED AT 400 WORDS)

The cytoskeleton of protozoan parasites

There are seven processes that require a cytoskeleton in protozoan parasites: nuclear division, cytokinesis, cell shape determination, motility, invasion, flagellar movement of sperm, and intracellular transport.

METHODS TO VISUALIZE ACTIN POLYMERIZATION ASSOCIATED WITH BACTERIAL INVASION

Publisher Summary This chapter discusses the methods to visualize actin polymerization associated with bacterial invasion. Because actin is the single most abundant protein in animal cells, it is not surprising that invading microorganisms must interact with it, at least indirectly. How close the association is depends on whether the microorganism is attached to the cell surface, resides in the cytoplasm enclosed in a phagosomal membrane, or enters the cytoplasm proper after breaking out of the phagosomal vacuole. Much has been learned about the interaction of intracellular pathogens and the host cell actin at the light microscope level by using fluorescently labeled molecules that bind to actin filaments in permeabilized cells, for example, phalloidin and phallicidin, or the injection of labeled actin or labeled actin-binding proteins into living cells. The latter techniques are particularly valuable as one can study living cells and assay by video microscopy exactly what the bacteria are doing. By control of the conditions and careful analysis of video sequences, what the bacteria are doing and how they are doing may be discerned.