Worms reveal essential functions for intermediate filaments

Intermediate filaments (IF) represent one of the three major cytoskeletal systems found in animal cells. Their somewhat uninspired name was originally derived from their 10-nm diameter, which lies between that of smaller actin-containing microfilaments and larger microtubules. This name, however, belies their importance as critical players in the organization of cells and tissues of vertebrate systems. As to function, it has become obvious from studies of numerous human disorders, such as those that cause blistering diseases of the skin, that IF play important roles in establishing and maintaining the mechanical integrity of cells (1). Depending on the cell type, IF proteins comprise anywhere from 1 to 85% of total cell protein and, despite these quantities, they remain the least studied and understood of all cytoskeletal systems. Historically, there are many reasons for this lack of understanding of their structure and function, the most obvious relates to the fact that the structural proteins that assemble into IF are not highly conserved. For example, humans contain IF that are encoded by over 50 different members of a multigene family. This family is subdivided into six types on the basis of similarities in their amino acid sequences (2). This is in stark contrast to the other cytoskeletal components whose core structures are comprised primarily of the highly conserved subunits of microtubules, α and β tubulin, and actin, the major subunit of microfilaments.

By probing the complete worm genome, there now appear to be only 11 genes encoding IF, which makes functional studies much more manageable relative to vertebrate systems.

There have been many attempts to use the gene knockout approach in the mouse to determine the function of specific types of IF. In some cases, this approach has provided insights into possible functions, but in other cases this approach has not revealed an obvious phenotype. In the case of the Type III desmin knockout mice, there are severe structural defects in cardiac muscles, demonstrating that IF play a critical role in establishing and maintaining the structural integrity of these cells (3). This is also the case for the keratin 8 knockout mouse, in which the majority of embryos die of internal bleeding because of the fragility of hepatocytes in day 12 embryos (4). However, in a different strain of mice, the same K8 knockout phenotype did not produce embryonic lethality. Postnatal examination of these mice showed only colorectal hyperplasia and an inflammatory response in the lamina propria and submucosa of the intestinal tract (5). Finally, in the case of two other Type III IF gene knockouts, no obvious phenotypes were initially discovered, and mice negative for vimentin (6) and glial fibrillary acidic protein (GFAP; ref. 7 and references therein) survived to reproductive age. However, more recent work has shown more subtle phenotypes in these mice, such as defects in wound healing in the vimentin knockouts (8), and in the response of astrocytes to traumatic brain injury in the GFAP null animals (7). From these studies, it is obvious that analyses of IF function in mouse models are extremely complex and difficult to interpret.

Determinations of IF functions are further complicated by the fact that the expression of different IF genes is temporally regulated during development. In many cases, progenitor cells express one type of IF and, as development proceeds, it is gradually replaced by another type of IF system. This often results in the coexpression of two types of IF in differentiating cells. For example, in the earliest stages of development, many cells (e.g., neuroblasts) destined to become neurons in the central nervous system express the Type III IF protein vimentin, and later during differentiation, vimentin is down-regulated as the Type IV “neurofilament triplet” proteins are expressed (9). An additional factor distinguishing IF from other cytoskeletal components is related to variations in the number of proteins required for their assembly. For example, in vitro studies have shown that the Type III IF proteins such as vimentin and desmin form homopolymer IF, whereas the Type I and II keratins can form IF only from heterodimers comprised of one of each type of protein chain, and Type IV neurofilaments require three protein chains, NFL, NFM, and NFH, to form proper IF (2). However, recent studies have revealed that most IF structures formed in vivo may be heteropolymers. For example, vimentin, which readily forms homopolymers in vitro, is frequently found as a heteropolymer with nestin, a Type VI IF protein in vivo. In vitro, nestin cannot assemble into IF on its own, but when mixed with vimentin, it combines to form typical IF (10, 11). Likewise, desmin is frequently associated with another Type VI protein, paranemin (12). The emerging picture from a large number of studies suggests that every distinct cell type has a different cytoskeletal IF composition. In addition, there is an ever-growing number of IF-associated proteins (IFAPs) such as plectin (13), which adds another level of complexity to this large family of proteins. Finally, recent studies using green fluorescent protein (GFP)-tagged IF have demonstrated that they are very dynamic and motile structures in vivo (14). Many of their motile properties have been linked to the activities of microtubule-associated motors such as kinesin (14). Taken together, all of these factors make studies of IF function even more challenging.

In this issue of PNAS, Karabinos et al. (15) take the first crack at testing IF function in a far less complicated and widely studied genetically approachable organism, Caenorhabditis elegans. By probing the complete worm genome, there now appear to be only 11 genes encoding IF, which makes functional studies much more manageable relative to vertebrate systems. On the basis of their amino acid sequence, these 11 genes can be subdivided into 5 subgroups: A (A1–A4), B (B1, B2), C (C1, C2), D (D1, D2), and E (E1). Furthermore, IF are present in cell types corresponding to the most extensively studied IF systems in vertebrate tissues, including epithelial, muscle, and nerve cells. Equally important is the fact that IF appear to bind to structures similar to hemidesmosomes in the hypodermis (epithelium), forming a complex involved in the transmission of forces from muscle to cuticle that are required for normal locomotion (16). In this study, the expression of each of the 11 C. elegans genes was suppressed or inhibited by using RNA interference (RNAi).

RNAi for each of these genes was injected into the gonads of hermaphrodite worms, and the resulting progeny were analyzed. Of the 11 genes, distinct phenotypes were observed for 5. These included A1–A3, B1, and C2. In the case of A1, development was normal until the L1 stage, but subsequently the larvae died within 3 to 6 days. Numerous physiological activities, including chemotaxis, locomotion, pharyngeal pumping, and overall morphology, appeared normal. However, the intestine appeared to be unusually swollen and contorted. In the majority of cases, larvae containing the A2 RNAi produced early larval lethality. These larvae exhibited abnormal positioning of their body musculature, abnormal locomotory behavior, and abnormal excretory canals (15).

A few of these A2 larvae matured a bit more, albeit slowly. However, development in these instances was arrested before the formation of the reproductive organs. These larvae were paralyzed except for a few very slow movements of the head and tail. In A3, ≈25% of the eggs never hatched. Those that did hatch showed severe abnormalities in body muscle organization, and the hypodermis did not attach properly to the cuticle.

Inhibition of B1 gene products was lethal in the later stages of development. A variety of abnormal phenotypes appeared, including “lumpy pretzels,” abnormal hypodermal morphologies, and abnormally short embryos in which the internal structures were compressed. For C2 gene product deficiencies, the animals displayed mild motility defects, with a small number also exhibiting a “dumpy morphology.” However, these worms did manage to develop into adults with reproductive capacity.

The expression patterns of some of the genes were also determined by Karabinos et al. by fusion with the GFP (15). Observations confirmed the expression of different IF genes at various developmental stages and in different types of cells. For example, A1 expression was seen in amphid sensory neurons, tail neurons, and some unidentified neurons. A1 was also expressed in the vulva, the pharyngeal–intestinal valve, and the rectum. A3 was found in the hypodermis during embryonic development and in larvae, but not in adults. This was also shown in a previous study using Northern blot analyses (17). For B1, expression was detected in amphid sensory and tail neurons, as well as cells in the excretory system, the uterus, vulva, rectum, pharyngeal-intestinal valve, and pharynx. This distribution of B1 was confirmed by immunofluorescence with a specific antibody. Overall, these results show that the members of two IF subgroups, A1 and B1, are expressed in a similar fashion, and that A3 is dramatically different.

From the results of this study, it is clear that four of the IF genes, A1–A3 and B1, are essential for the normal development of C. elegans. Karabinos et al. note, however, that it has been shown that some genes are not susceptible to the inhibitory effects of RNAi, and that this technique may be less effective in the inhibition of genes expressed in later stages of development. Therefore, they leave the door open for determining whether the six IF types unaffected by this approach are, or are not, essential for producing normal worms. Future studies using different methods will be required before conclusions can be drawn regarding the entire family of worm IF proteins.

Observations confirmed the expression of different IF genes at various developmental stages and in different types of cells.

Karabinos et al. (15) also speculate on the IF-related mechanisms underlying the paralysis exhibited by the larvae that develop after exposure to A2 and A3 RNAi. These larvae are paralyzed, and their muscles are abnormally organized. This could be because of a loss of transmission of force generated by the muscle cells to the cuticle via the hypodermis. Normally, force is transmitted to the cuticle through connections that include muscle cell dense bodies, the basal lamina, and the hypodermis. Within the hypodermis, hemidesmosomes located at both basal and apical surfaces, are connected to IF. Therefore a lack of IF in the hypodermis could easily alter the structural integrity of hemidesmosomes and the interactions required for force transduction to the cuticle, thereby preventing normal locomotion. In further support of the role of IF in maintaining the integrity of hemidesmosomes, A3 inhibition also results in the detachment of the hypodermis from the cuticle.

It should be noted that there is a single nuclear lamin gene in C. elegans. The lamins have been designated the Type V IF proteins. These nuclear targeted IF proteins assemble in the nucleus to become the major constituent of the nuclear lamina, which lies at the interface between the inner nuclear envelope membrane and chromatin. The lamina is thought to be important in establishing and maintaining nuclear shape and the interphase organization of chromatin. It has also been shown that the lamins form intranuclear structures involved in DNA replication (18). In C. elegans, the lamin gene, lmn-1, is responsible for the production of the Ce-lamin protein. Embryonic lethality results when the production of this protein is inhibited by using the RNAi technique (19), although some abnormal nuclei are formed during the early stages of development. These abnormalities include changes in interphase nuclear morphology, loss of chromosomes, abnormal condensation of chromatin, abnormal segregation of chromosomes, and aberrant nuclear pore formation (19). In Drosophila, where no cytoplasmic IF genes have been found, there are two nuclear lamin genes, DM₀ and C. Genetic analyses have shown that DM₀ is essential for nuclear organization, and mutations in this gene result in the formation of abnormal nuclear envelopes, nuclear pore clustering, and the accumulation of annulate lamellae, the storage forms of nuclear envelopes. This produces severe defects, including slow developmental progression, sterility, and impaired locomotion (20).

Finally, Karabinos et al. (15) make a very interesting point regarding the lack of cytoskeletal IF genes in the Drosophila genome. Essentially, they ask how flies can compensate for this lack of IF. The answer may lie in the way they organize their microtubules. In this regard, ultrastructural studies of arthropods suggest that microtubule arrays may provide the structural roles that IF carry out in vertebrate cells. For example, in fruit fly wing epidermal cells there are bundles of up to 900 microtubules traversing the cytoplasm, which are thought to be critical for maintaining the mechanical integrity of these cells (21). An interesting and perhaps related finding is that mice with reduced numbers of neurofilaments show an increase in the number of microtubules in large axons (22). It is therefore possible that even in vertebrates, microtubules can compensate for a loss of cytoskeletal IF.

In summary, the results of the studies described by Karabinos et al. (15) make it clear that cytoskeletal IF play essential roles in the development of C. elegans. Future studies of this worm may take a more genetic approach and provide even greater insights into the functions of this cytoskeletal system. These insights might be critical for furthering our knowledge of the larger families of IF in vertebrate organisms including humans. This is extremely important, as a large number of human diseases have been associated with abnormalities in IF cytoskeletal systems (1).