fat facets
Eukaryotic genomes encode large families of deubiquitinating enzymes (DUBs). Genetic data suggest that Fat facets (Faf), a Drosophila DUB essential for patterning the compound eye, might have a novel regulatory function; Faf might reverse the ubiquitination of a specific substrate, thereby preventing
proteasomal degradation of that protein. Additional genetic data implicate Liquid facets (Lqf), a homolog of the vertebrate endocytic protein epsin, as a candidate for the key substrate of Faf. Here, biochemical
experiments critical to testing this model were performed. The results show definitively that Lqf is the key substrate of Faf in the eye; Lqf
concentration is Faf-dependent, Lqf is ubiquitinated in vivo and deubiquitinated by Faf, and Lqf and Faf interact physically (Chen, 2002).
To detect Lqf protein levels in developing eyes, an
antibody was generated to Lqf. Eye discs were double-labeled with anti-Lqf and antibodies to the endocytic protein Shibire. This shows that Lqf and Shi colocalize at cell membranes: Lqf and Shi are concentrated apically in
cells within the morphogenetic furrow, an indentation that marks the
onset of differentiation, and also in developing
photoreceptors where their membranes meet. Similar results were obtained with
antibodies to two other endocytic proteins (Dap160 and alpha-Adaptin
[alpha-Ada]), and with phalloidin, which labels f-actin at cell
membranes (Chen, 2002).
One prediction of the hypothesis that Faf activity prevents the
degradation of Lqf is that in the developing eyes (larval eye discs) of
faf null mutant flies, there should be less Lqf protein than
in wild-type eyes. It was expected there would be less Lqf protein, as opposed
to no Lqf protein, because the lqf null mutant eye phenotype
is much more severe than the faf null mutant eye phenotype (Chen, 2002).
To test whether the level of Lqf is affected by faf+
gene activity, first the levels of Lqf were compared in adjacent groups of faf+ and
faf- cells in the eye disc, using confocal microscopy. Clones of
homozygous faf- cells were generated in faf+/faf- heterozygous eye discs,
marked by the absence of ß-galactosidase (ß-gal) expression. The eye discs containing clones were triple-labeled with antibodies to ß-gal (to outline the clones), to
Lqf (to detect the level of Lqf protein), and to Shi (as a negative
control). It was found that throughout the eye disc, the level of Lqf
protein, reflected in the strength of the signal from antibody
labeling, is lower within the faf- clones than in
the faf+/faf- heterozygous cells
surrounding them. In contrast, the levels of Shi
protein are the same within and outside the clone boundaries (Chen, 2002).
To quantify the difference in Lqf protein levels in
faf+ and faf- cells, the levels of Lqf were assayed in eye disc protein extracts prepared from wild-type
and faf- flies in Western blot experiments. Homozygotes for two different mutant faf alleles that behave genetically as strong
loss-of-function mutations were used: fafBX4 is an inversion
that makes no functional Faf protein, and fafFO8
encodes an Faf protein with histidine residue 1986, which is critical
for DUB catalytic activity, changed to tyrosine. There is
two- to three-fold less Lqf in eye disc protein extracts of
faf- homozygotes than in wild-type extracts. faf+ transgene restores function back to faf- flies. The transgene containing faf+ genomic DNA, which when introduced into faf- homozygotes complements the mutant eye
phenotype, results in a two- to three-fold increase in Lqf protein level in eye disc extracts. A nearly identical transgene that fails to complement the
faf- mutant phenotype because it has a point
mutation in the codon for cysteine 1677, which is critical to the DUB
activity of Faf, fails also to increase the level of Lqf protein in eye disc extracts. It is concluded that faf+ activity results in an increase in the level of Lqf protein (Chen, 2002).
A second prediction of the model wherein Faf prevents proteolysis
of Lqf by deubiquitinating it, is that there should be Lqf protein
linked to Ub chains present in eye discs. Ubiquitinated proteins are
usually detected on Western blots as ladders of protein bands of higher
molecular weight than the protein in question, in increments of ~8
kD; each 'rung' on the ladder represents a protein species with a
Ub chain that is one Ub residue longer than the previous rung. Proteins
with Ub chains are rapidly degraded, and thus difficult to detect;
usually, inhibition of proteasome and/or DUB activity is required to
detect them. It this study, inhibition of the DUB activity of Faf, genetically, stabilizes ubiquitinated forms of Lqf (Chen, 2002).
It is concluded that in eye discs, Lqf is ubiquitinated, and subsequently
either deubiquitinated by Faf or degraded. The observation that
considerable amounts of nonubiquitinated Lqf protein remain in
faf- eye discs indicates either that only a
fraction of the Lqf protein in the eye disc is ubiquitinated, and/or
that DUBs other than Faf also deubiquitinate some Lqf protein (Chen, 2002).
A third prediction of the model wherein Lqf is the substrate of
Faf is that the proteins should, either directly or indirectly, interact. Anti-Lqf was used to immunoprecipitate Lqf from protein extracts prepared from embryos, and tested for the presence of Faf in
the immunoprecipitates on Western blots.
Embryos were used because sufficient protein could not be obtained from
eye discs. In addition, to facilitate detection of Faf, the embryos
were transformed with a P{hs-myc-faf+}
transgene, which expresses a fully functional, myc-tagged Faf protein
upon heat shock, that can be detected on Western blots with anti-myc. myc-Faf was detected in the anti-Lqf immunoprecipitate of the protein extract from
heat-shocked transformant embryos (Chen, 2002).
It is concluded that myc-Faf and endogenous Lqf proteins interact
physically in Drosophila embryos. Bacterially produced or in
vitro translated partial Faf and full-length Lqf proteins do not bind
to each other in GST pull-down assays. One possible explanation is that only full-length Faf can bind to Lqf in these assays. Alternatively, Faf and Lqf may require other proteins for their interaction (Chen, 2002).
These experiments provide critical biochemical evidence for a model in which a DUB called Faf specifically deubiquitinates Lqf protein, thereby preventing its proteolysis. There is less Lqf protein in the developing eye in the absence of catalytically functional Faf protein, that Lqf is ubiquitinated and subsequently deubiquitinated by Faf, and that Faf and Lqf interact physically. Taken together with previous genetic evidence that provides strong support for the model, it is concluded that Faf is a substrate-specific regulator of ubiquitination, a novel function for a DUB (Chen, 2002).
The eyes of faf null or lqf hypomorphic mutants have
more than the normal complement of eight photoreceptor cells in each facet, owing to the failure of a cell communication pathway early in
eye development. The Faf/Lqf interaction is essential in only a
small number of cells in the eye disc, which must be particularly
sensitive to the levels of Lqf, and in these cells, Lqf presumably
controls the frequency or specificity of endocytosis. Although the
precise mechanism of epsin function is unknown, vertebrate epsin binds
to the endocytosis complex and also to PIP2 at the cell
membrane, and is required for endocytosis (Chen, 1998; Itoh, 2001). Apparently, appropriate endocytosis in this small group of
cells is essential for successful communication with their neighbors;
increased Lqf levels either enables these cells to send a signal to
their neighbors that inhibits neural determination, or else prevents
them from sending their neighbors a positive differentiation signal (Chen, 2002).
Through a variety of mechanisms, endocytosis is proposed to regulate
ligand/receptor interactions during development. How Lqf and endocytosis regulate faf+-dependent cell signaling remains to be
determined. Since faf has vertebrate homologs, this mode of regulation is likely to be conserved. The finding that Lqf is the key substrate of Faf in the Drosophila eye shows not only that a DUB can regulate
ubiquitination and thus proteolysis, but also that an endocytosis
complex protein can be a target for the control of a cell communication
event critical to cell determination (Chen, 2002).
The Fat facets protein
(FAF) acts as a regulatory Ubiquitin-specific protease (Ubp) that prevents degradation of its substrate by the proteasome. Flies
bearing fat facets gene mutations have been used to show that a Ubp is a cell type- and substrate-specific regulator of cell fate decisions. Ubiquitin conjugates are cleaved by a 744-amino acid partial Faf protein that includes the Cys and His domains. Four mutant faf genes that encode proteins with either an altered key Cys residue or with changes in one or both of the two conserved His residues abolish the Udps activity of Fas (Huang, 1995).
Anterior-posterior patterning and germ cell specification in Drosophila requires the establishment, during oogenesis, of a specialized cytoplasmic region termed the pole plasm. Numerous RNAs and proteins accumulate to the pole plasm and assemble in polar granules. Translation of some of these RNAs is generally repressed and active only in pole plasm. Vasa (Vas) protein, an RNA helicase and a component of polar granules, is essential maternally for posterior patterning and germ cell specification, and Vas is a candidate translational activator in the pole plasm. Vas is stabilized within the pole plasm in that it is initially present throughout the entire embryo but strictly limited to the pole cells by the cellular blastoderm stage. hsp83 mRNA, which accumulates in the pole plasm through a stabilization-degradation mechanism, is another example. A biochemical approach has been used to identify proteins that copurify with Vas in crosslinked extracts. Prominent among these proteins was the ubiquitin-specific protease Fat facets (Faf), a pole plasm component, but one whose roles in posterior patterning and germ line specification have remained unclear. Evidence suggests that Faf interacts with Vas physically and reverses Vas ubiquitination, thereby stabilizing Vas in the pole plasm (Liu, 2003).
Vas and Faf can be copurified from chemically crosslinked embryonic and ovarian extracts; in faf mutants, the ubiquitination of Vas is increased and its levels are decreased in ovaries and more strikingly in progeny embryos. The simplest interpretation of these data is that Vas is a specific substrate for the deubiquinating enzyme Faf. It is believed the reduction of localized Osk observed in faf mutant ovaries is an indirect result of the reduced stability of Vas, since vas function is required for the stable accumulation of Osk in the pole plasm (Liu, 2003).
Heterozygotes for a vas null mutation produce embryos with a 20%-25% reduction in pole cell number, yet such embryos, unlike progeny of faf mutant mothers, show prominent posterior Vas staining. The severe phenotype of embryos produced by homozygous faf mothers renders difficult a direct analysis of its requirement in the pole plasm. However, based on the vas heterozygous phenotype, the obvious reduction in posterior Vas accumulation in faf mutants should affect development, and therefore Faf-mediated stabilization of Vas must contribute to pole plasm function. Faf-mediated protection of Vas is mostly restricted to the pole plasm, perhaps because during oogenesis, Faf itself becomes localized to the posterior pole and is incorporated into pole cells. As is the case for other pole plasm components, Faf localization depends on osk function, and a role for Faf in germ cell differentiation and development has been proposed. Interestingly, Usp9x, a bona fide mouse ortholog of Faf, is predominantly expressed in both germ cell and supporting cell lineages during gonadal development, suggesting a conserved role for Faf in mammalian germ line development (Liu, 2003).
Long-distance organelle transport toward axon terminals, critical for neuron development and function, is driven along microtubules by kinesins . The biophysics of force production by various kinesins is known in detail. However, the mechanisms of in vivo transport processes are poorly understood because little is known about how motor-cargo linkages are controlled. A c-Jun N-terminal kinase (JNK)-interacting protein (JIP1) has been identified previously as a linker between kinesin-1 and certain vesicle membrane proteins, such as Alzheimer's APP protein and a reelin receptor ApoER2. JIPs are also known to be scaffolding proteins for JNK pathway kinases. Evidence is presented that a Drosophila ubiquitin-specific hydrolase (Fat facets) and a JNK signaling pathway that it modulates can regulate a JIP1-kinesin linkage. The JNK pathway includes a MAPKKK (Wallenda/DLK), a MAPKK (Hemipterous/MKK7), and the Drosophila JNK homolog Basket. Genetic tests indicate that those kinases are required for normal axonal transport. Biochemical tests show that activation of Wallenda (DLK) and Hemipterous (MKK7) disrupts binding between kinesin-1 and APLIP1, which is the Drosophila JIP1 homolog. This suggests a control mechanism in which an activated JNK pathway influences axonal transport by functioning as a kinesin-cargo dissociation factor (Horiuchi, 2007).
Maintaining proper distributions of protein complexes, RNAs, vesicles, and other organelles in axons is critical for the development, function, and survival of neurons. The primary distribution mechanism relies on long-distance transport driven by microtubule motor proteins. Components newly synthesized in the cell body, but needed in the axon, bind kinesin motors that carry them toward microtubule plus ends and the axon terminal (anterograde transport). Neurotrophic signals and endosomes, examples of axonal components that require transport to the cell body, bind dynein motors that carry them toward minus ends (retrograde transport). The importance of these processes is highlighted by the observation that mutation of motors and other transport machinery components can cause neurodegenerative diseases in humans and analogous phenotypes in model organisms (Horiuchi, 2007).
Two key questions are (1) how do cargoes link to particular motors, and (2) how are such linkages regulated to ensure appropriate pickup and dropoff dynamics? For kinesin-vesicle linkages, scaffolding proteins have emerged as key connectors. For example, the cargo-binding kinesin light chain (Klc) subunit of kinesin-1 binds not only the kinesin-1 heavy chain (Khc) but also JNK-interacting proteins (JIPs). Vertebrate JIPs can bind multiple components of the JNK signaling pathway, e.g., JNK itself, upstream activating kinases (MAPKKs), and regulatory kinases (MAPKKKs). JIPs can also bind vesicle-associated membrane proteins, such as ApoER2, which is a reelin receptor, and APP, a key factor in Alzheimer's disease. Therefore, JIP scaffolding proteins are likely to link JNK pathway kinases and kinesin-1 to vesicles carrying these membrane proteins. This raises an interesting question: Are the JNK pathway kinases simply passive hitchhikers on the kinesin-1/JIP/vesicle complex, or can they actively regulate its transport (Horiuchi, 2007)?
A genetic screen was conducted for factors that control kinesin-JIP linkage during axonal transport. The screen was based on the previous observation that neuron-specific overexpression of Aplip1, which encodes the Drosophila JIP1, causes synaptic protein accumulation in axons, larval paralysis, and larval-pupal lethality, the classic axonal-transport-disruption phenotypes caused by Khc and Klc mutations. Why might overexpression of the JIP1 cargo linker for kinesin-1 disrupt axonal transport? The disruptive effect requires APLIP1 (JIP1)-Klc binding. It may be that excess APLIP1 (JIP1) competes with other Klc-binding proteins, for example, different linkers that may attach kinesin-1 to other cargoes. In search of factors that can disrupt or antagonize APLIP1 (JIP1)-Klc binding, a screen was performed for genes that can suppress the axonal-transport phenotypes when co-overexpressed with Aplip1. An 'EP' collection of fly strains capable of the targeted overexpression of endogenous Drosophila genes was screened and P{EP}fafEP381, a line that overexpresses fat facets (faf), was identified as a strong suppressor of the APLIP1 (JIP1)-induced lethality and other neuronal overexpression phenotypes (Horiuchi, 2007).
Faf protein antagonizes ubiquitination and proteasome-mediated degradation of its target proteins. Interestingly, Faf was recently reported to stimulate a Drosophila neuronal JNK signaling pathway that is regulated by the MAPKKK Wallenda (Wnd), a homolog of dual leucine zipper-bearing kinase (DLK) that is known to bind JIP1. Overexpression of faf leads to increased levels of Wnd (MAPKKK) protein and thereby causes excessive synaptic sprouting through a pathway that requires the Drosophila JNK homolog Basket. It was found that mutating just one copy of wnd blocked the suppression of Aplip1 overexpression by P{EP}fafEP381. This suggests that faf overexpression suppresses APLIP1 (JIP1)-Klc interaction by elevating the level of Wnd (MAPKKK). Consistent with this, direct overexpression of wnd in neurons with a wild-type transgene (UAS-wnd) was as effective as P{EP}fafEP381 in suppressing UAS-Aplip1-induced axonal accumulation of synaptic proteins. Equivalent expression of a 'kinase-dead' mutant transgene (UAS-wndKD) did not suppress the defects. Thus, Wnd (MAPKKK) and its downstream phosphorylation targets may actively regulate APLIP1 (JIP1)-Klc binding in neurons (Horiuchi, 2007).
See the embryonic expression pattern of faf at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
Staining in the eye disc reveals protein within the entire region ahead of the morphogenetic furrow, including the antennal disc, and lower levels behind the furrow. Protein is also consistently detected in other discs and larval and adult tissues (i.e. fat body, gut, larval ovary and testes and adult male sex organs) (Fischer-Vize, 1992).
Faf protein is seen throughout oogenesis within the nurse cell-oocyte cluster. When the oocyte becomes distinct from the nurse cells, cytoplasmic stain appears to be confined to the nurse cells. Later, high levels of stain are seen at the posterior pole of oocytes. The posterior staining persists throughout oogenesis, and after the egg is laid, maternal protein at the posterior becomes incorporated into the pole cells. FAF mRNA is not posteriorly localized. Faf protein is also found in testis (Fischer-Vize, 1992).
Localization of Faf protein depends on Oskar. As Staufen protein and OSK mRNA are posteriorly localized earlier than Faf protein, and Vasa protein is localized at about the same time as Faf, faf appears to fit into the posterior group hierarchy. Pole plasm components are posteriorly localized in a normal fashion in faf mutants and Nanos protein function is unimpaired in faf mutant embryos (Fischer-Vize, 1992).
All the strong faf alleles cause female sterility. Females homozygous for any strong allele have apparently normal ovaries, but lay eggs that never form cuticle and never hatch. In embryos from faf mutant mothers, no normal syncytial blastoderm embryos are observed. Most of the nuclei fail to migrate to the periphery. Also commonly observed are embryos in which patches of asynchronously dividing nuclei have migrated to the periphery. Except for the pole cells (which are fewer in number and spread out, instead of grouped together as in wild type), no cellularization of faf mutant embryos is ever observed (Fischer-Vize, 1992).
Many defects are present in strong faf mutant retinas, the most striking of which is the appearance of one, two or occasionally three outer photoreceptor cells in most facets (Fischer-Vize, 1992).
In order to identify the genes encoding the
substrate of Fat facets and other components of the neural inhibition pathway, a mutagenesis
screen for dominant enhancers of the fat facets mutant eye phenotype was performed. Several genes
were identified, one of which is an excellent candidate for encoding a component of the pathway
regulated by Fat facets. Three different eye phenotypes were observed when the fat facets
mutants were dominantly enhanced by different mutations, suggesting that fat facets has other
functions in addition to its critical role early in eye development (Fischer, 1997).
The Drosophila fat facets (faf) gene encodes a deubiquitination enzyme with a putative function in proteasomal protein degradation. Mutants lacking zygotic faf function develop to adulthood, but have rough eyes caused by the presence of one to two ectopic outer photoreceptors per ommatidium. faf interacts genetically with the receptor tyrosine kinase (RTK)/Ras pathway, which induces photoreceptor differentiation in the developing eye.
faf also interacts with pointed. The extra-photoreceptor phenotype observed in faf mutants is clearly suppressed by pointed mutation; many more ommatidia have six outer photoreceptors in a trapezoidal arrangement characteristic of wildtype ommatidia. yan mutation in combination with faf strongly enhances the faf phenotype. Reducing the D-Jun activity suppresses the faf mutant phenotype. In sevenless;faf double mutants, R7 cells, normally absent in sevenless mutants, form in 60% of the ommatidia. Thus, faf can alleviate the requirement for sev in the R7 precursor. These results indicate that RTK/Ras signaling is increased in faf mutants, causing normally non-neuronal cells to adopt photoreceptor fate. Consistently, the protein level of at least one component of the Ras signal transduction pathway, the transcription factor D-Jun, is elevated in faf mutant eye discs when the ectopic photoreceptors are induced. It is proposed that defective ubiquitin-dependent proteolysis leads to increased and prolonged D-Jun expression, which together with other factors contribute to the induction of ectopic photoreceptors in faf mutants. These studies demonstrate the relevance of ubiquitin-dependent protein degradation in the regulation of RTK/Ras signal transduction in an intact organism (Isaksson, 1997).
The Drosophila fat facets gene encodes a deubiquitinating enzyme required during eye development to limit to eight the
number of photoreceptors in each facet. Ubiquitin is a small polypeptide that targets proteins for degradation
by the proteasome. Deubiquitinating enzymes cleave ubiquitin-protein bonds. In order to investigate the role of Fat facets in the ubiquitin pathway, genetic interactions between fat facets and the Drosophila UbcD1 gene were
assessed. In addition, three yeast deubiquitinating enzyme genes were tested for their ability to substitute for fat facets
in the developing Drosophila eye and for their effects on eye morphology. These experiments support
the hypothesis that Fat facets activity antagonizes that of the proteolytic machinery (Wu, 1999).
Fat facets is a deubiquitinating enzyme required in a cell
communication pathway that limits to eight the number
of photoreceptor cells in each facet of the Drososphila
compound eye. Genetic data support a model whereby
Faf removes ubiquitin, a polypeptide tag for protein
degradation, from a specific ubiquitinated protein, thus
preventing its degradation. Mutations in the liquid facets gene have been identified as dominant enhancers of the
fat facets mutant eye phenotype. The liquid facets locus
encodes epsin, a vertebrate protein associated with the
clathrin endocytosis complex. Genetic
experiments reveal that fat facets and liquid facets facilitate
endocytosis and function in common cells to generate an
inhibitory signal that prevents ectopic photoreceptor
determination. The fat facets mutant phenotype is extraordinarily sensitive to the
level of liquid facets expression. It is proposed that Liquid
facets is a candidate for the critical substrate of Fat facets
in the eye (Cadavid, 2000).
There are three key components of the endocytosis complex: (1) clathrin, which forms a cage structure
engulfing the cell membrane; (2) AP-2, the core adaptor
complex, which binds to clathrin and brings it to the cell
surface and (3) dynamin, a GTPase required for vesicle
formation. Additional proteins associated with AP-2 have been
identified, many of which contain protein-protein interaction
domains called EH-domains and EH-domain-binding motifs. Epsin is an EH-domain-binding protein identified as a
partner for Eps15, an EH-domain protein
that also binds AP-2. The large number of AP-2-binding proteins
identified suggests that many of them may have temporal
and/or tissue-specific functions (Marsh and McMahon, 1999).
The precise roles of Eps15 and epsin in endocytosis are
unknown (Cadavid, 2000 and references therein).
The lqf gene itself is essential in Drosophila; in an otherwise
wild-type background, lqf null mutants
die as embryos. Clones of cells in the eye in which there is
little or no lqf gene function have severely disrupted eye
morphology indicating that lqf is required
also after embryogenesis for eye development.
The mutant phenotypes associated with two weak lqf mutant
alleles reveal specific roles for
lqf in eye, wing and leg development (Cadavid, 2000).
The eye defects in
homozygous adults for a weak allele of lqf resemble those in faf null mutants. As in faf mutants,
the additional photoreceptors in lqf mutants arise from specific
precursor cells (M-cells) present early during eye development. In contrast to lqf null mutants, faf null mutants are
viable, have normal wings and legs and have less severe eye
defects. Thus, lqf functions more
broadly than faf, but both the lqf and faf genes are required
during eye development in order to prevent the M-cells from
becoming photoreceptors (Cadavid, 2000).
The faf mutant eye phenotype is unusually sensitive to a
decrease in the dose of the lqf gene, suggesting strongly that the
two genes function in a common pathway. Genetic interactions
with endocytosis and Ub pathway mutants show that faf and lqf
facilitate endocytosis and antagonize ubiquitination. In
addition, although lqf is more broadly required than faf in the
eye and elsewhere in the fly, weak lqf mutations reveal that like
faf, lqf is required to prevent the misdetermination of M-cells
as photoreceptors. Moreover, when expressed only in the rough positive
cells surrounding the facet preclusters, both faf and lqf genes
rescue completely to wild-type their respective M-cell
misdetermination mutant phenotypes in the eye. Finally, given
the relationship between Faf and its substrate protein, it would
be expected that increasing the dose of the substrate should
suppress the faf mutant phenotype. Slight
overexpression of lqf completely obviates the need for faf in eye
development. The simplest model consistent with all of this
genetic data is that Lqf is the substrate of Faf. Other more
complicated explanations are, of course, possible (Cadavid, 2000).
There is biochemical evidence that AF-6, a scaffolding protein
thought to modulate cell-cell junctions in response to Ras
activation may be an in vivo substrate of Fam, the mouse homolog
of Faf; AF-6 and Fam bind each other in
vitro and ubiquitinated AF-6 can be detected and
deubiquitinated by Fam in cultured cells.
Like lqf, the Drosophila Af6 homolog, canoe, is required
pleiotropically for Drosophila eye development. In contrast to lqf mutations, however, canoe
mutations do not act as strong dominant enhancers of the faf
mutant eye phenotype . Given
the striking genetic interactions between faf and lqf, it seems
that canoe is unlikely to play a significant role in the essential
faf pathway in the eye (Cadavid, 2000 and references therein).
While only one Faf/substrate interaction may be essential to
normal eye development in Drosophila, Faf and Fam may have
several substrates in vivo. Normally non-essential roles for faf
later in eye development have been revealed in particular
mutant backgrounds and
Faf could have different substrates for its critical role in M-cell
fate determination than in its redundant roles. Moreover, in
addition to its essential role in eye development, faf is required
maternally for cellularization of embryos and the critical maternal substrate of Faf is unknown.
Because faf has mouse and human
homologs, the modes of regulation by Faf are likely to be
conserved. However, it is possible that the critical substrate(s)
of Faf in Drosophila may differ from those in vertebrates (Cadavid, 2000 and references therein).
If Lqf is the substrate of Faf, then epsin levels, determined by
the balance between its ubiquitination and deubiquitination,
could regulate endocytosis. Mono-ubiquitination, however, has
been shown previously to regulate endocytosis in two different
ways. (1) Mono-ubiquitination of cell surface receptors can
act as a signal for receptor endocytosis, which leads to
lysosomal degradation. Here, the Ub moiety is somehow recognized by the
endocytosis machinery; this process has nothing to do with the
proteasome. (2) Eps15, an endocytosis complex
component in mammalian cells, is mono-ubiquitinated in response
to EGF receptor activation and Eps15 may require this
modification to stimulate receptor endocytosis. In addition, Pan1p a yeast protein similar to Eps15, is
required for endocytosis in yeast. Although it is unknown whether Pan1p
is mono-ubiquitinated in yeast, there is evidence that
ubiquitination of an endocytic complex component is required
for endocytosis in yeast; Rsp5p, a component of the
ubiquitination machinery called a ubiquitin-ligase, may bind to
Pan1p and is required generally for endocytosis in yeast, even
for endocytosis of proteins with non-Ub endocytosis signals (Cadavid, 2000 and references therein).
Since Eps15 binds to epsin, could a mono-ubiquitinated
Drosophila Eps15 homolog be the substrate of
Faf? Two of experimental results are inconsistent with this
model. (1) It has been shown previously that the activity of
Faf antagonizes proteolysis, not just
ubiquitination; mutations in a gene encoding
a proteasome subunit act as strong suppressors of the faf mutant
eye phenotype. This result strongly suggests that Faf activity
antagonizes proteolysis and thus that Faf deubiquitinates a
protein containing a Ub chain targeting it for degradation,
rather than a mono-ubiquitinated protein. (2) If mono-ubiquitination
of Eps15 activates it, as the available data
suggests, then deubiquitination of Eps15 by Faf would render
Eps15 inactive and thus the function of Faf would antagonize
endocytosis. The data presented here clearly indicate the
opposite; mutations in endocytosis complex genes (particularly
lqf and Clathrin heavy chain) act as strong dominant enhancers of faf, suggesting
that the normal function of Faf is to facilitate endocytosis (Cadavid, 2000 and references therein).
Elevated levels of Lqf obviate the need for Faf, presumably by
stimulating epsin-dependent endocytosis generally or
stimulating endocytosis of a specific cell surface protein. How
can the observation that Lqf and Faf function outside the M-cells
to determine M-cell fate be reconciled with a role for Lqf
in endocytosis? Endocytosis is known to modulate ligand/receptor interactions by a variety of mechanisms. One
possibility is that M-cell fate is affected by a diffusible ligand
that, like Wingless, travels
via endocytosis through several cell distances. Alternatively,
regulation of a membrane-bound receptor by endocytosis in
cells adjacent to the M-cells could affect M-cell fate indirectly.
For example, EGF receptor activity is downregulated by
endocytosis following ligand binding. By contrast, activity of the
Notch receptor may be up-regulated by endocytosis of
activated receptors whose intracellular domains have been
cleaved off prior to their translocation into the nucleus.
Membrane-bound Notch receptors lacking their intracellular
domains display dominant negative activity and endocytosis of cleaved Notch
receptors may be required normally for precise modulation of
Notch activity. Patterning of the
photoreceptor preclusters in the developing eye may require
that both Notch and the EGF receptor are activated in the rough-expressing
cells surrounding the facet preclusters. Thus, Faf
could regulate the activity of one or both of these receptors (Cadavid, 2000 and references therein).
The Drosophila DNAprim gene encodes the large subunit (60 kD) of DNA primase, the part of DNA polymerase alpha that synthesizes RNA primers during DNA replication. The precise function of the 60-kD subunit is unknown. In a mutagenesis screen for suppressors of the fat facets (faf) mutant eye phenotype, mutations in DNAprim were identified. The faf gene encodes a deubiquitinating enzyme required specifically for patterning the compound eye. The DNA sequences of four DNAprim alleles have been determined and these define essential protein domains. While flies lacking DNAprim activity are lethal, flies with reduced DNAprim activity display morphological defects in their eyes, and unlike faf mutants, cell cycle abnormalities in larval eye discs (Chen, 2000b).
Eight of the 11 DNAprim alleles, including In(3L)78Cb1, are early lethal in trans to Dfr(3L)rdgC-co2 or S(faf)240. Since In(3L)78Cb1 breaks within the coding region of DNAprim, the other 7 alleles are also likely to be null mutants. A weaker allele, DNAprimT2, is pupal lethal in trans to Dfr(3L)rdgC-co2 or S(faf)240. There is cell death in the eye anterior of late-stage pupae of these genotypes. The weakest alleles are the homozygous viable group of imprecise excisions, exemplified by DNAprimj10B2Delta13-15. When homozygous or in trans to Df(3L)rdgC-co2, the DNAprimj10B2Delta13-15 alleles result in slightly roughened external eyes. Patterning defects, either in the accessory cells (cone and pigment cells) of pupal eyes, could not be detected in these flies. Thus, the external rough eyes are most likely due to subtle defects in cone cells, pigment cells, and/or bristles in the final stages of eye development; absent bristles and bristle misplacements are apparent in the adult eyes (Chen, 2000b).
To understand the basis for the genetic interactions observed between faf and DNAprim, it was asked whether the cell cycle is altered in these mutants. DNAprim mutants would be expected to have difficulty during S phase when DNA is replicated. If S phase is slower in these mutants, then more cells would be expected to be in S phase at any given time. During larval eye disc development, undifferentiated cells anterior to the morphogenetic furrow divide asynchronously and then arrest in G1 within the morphogenetic furrow prior to differentiation. No cell division occurs again until later in the eye disc, when cells that will give rise to three of the eight photoreceptor types and all of the other cell types undergo a single mitosis. By assaying BrdU incorporation with anti-BrdU antibodies, cells in S phase were visualized in larval eye discs of flies with a pupal lethal combination of DNAprim mutations, as well as wild-type and faf - eye discs. BrdU incorporation in the faf - eye discs resembles wild type. By contrast, in the DNAprim mutants, more cells are in S phase at a given time. Since faf mutants do not have defects in S phase, the genetic interactions between DNAprim and faf most likely reflect indirect interactions between the functions of the two proteins (Chen, 2000b).
Why do DNAprim mutations suppress the faf mutant eye phenotype? To answer this question, the origins of the ectopic photoreceptors in faf mutant eyes must be considered and also the time and place in the larval eye disc in which faf normally functions. The ommatidia of faf mutant eyes contain one or more photoreceptors in addition to the normal eight. The extra photoreceptors originate from the mystery cells, which are present in early facet preclusters. The precursors to the first five photoreceptors (R8, R2/5, and R3/4) and one or more mystery cells emerge posterior to the morphogenetic furrow as preclusters of six or more cells, each surrounded by undifferentiated cells. Normally, the mystery cells detach from the preclusters and go on to divide in the wave of mitosis posterior to the furrow, giving rise to the remainder of the photoreceptors (R1, R6, and R7) and to the accessory cells (cone, pigment, and bristle cells). In faf mutants, the mystery cells remain within the preclusters and become extra R3/4-like cells. Although faf is required for inhibition of mystery cell neurogenesis, mosaic analysis as well as faf transgene complementation experiments indicate that faf is not required within the mystery cells nor within any photoreceptor precursors in the precluster (Chen, 2000b).
Normally, faf is expressed at high levels anterior to the morphogenetic furrow, where cells are undergoing mitosis, and then its expression shuts off within the furrow, just prior to precluster formation. Although faf normally functions anterior to the furrow, later expression of faf in a subgroup of cells within the furrow is sufficient to rescue the faf mutant eye phenotype. The subgroup of cells that require faf expression are those cells that express the rough gene; these cells surround those that express the proneural gene atonal, which are destined to become the precluster cells. Thus, faf activity in the dividing cells anterior to the furrow facilitates a cell communication pathway that may occur anterior to or within the furrow. Perhaps when DNA primase levels are halved in heterozygous mutants, the cells anterior to the furrow remain in S phase longer, which changes subtly the output of the signaling pathway that faf modulates in such a way that the requirement for faf is largely overcome (Chen, 2000b).
Mutations in cell cycle proteins have been isolated previously in genetic screens for mutants affecting the Sevenless receptor tyrosine kinase signaling pathway in eye development. For example, mutations in the Drosophila homolog of Saccharomyces cerevisiae cell cycle regulator cdc37 were identified as dominant enhancers of weak sevenless mutants. Also, mutations in the peanut gene, which encodes a protein similar to yeast bud neck filaments and functions in cytokinesis, were identified as dominant enhancers of the phenotype of weak seven in absentia mutants; seven in absentia is a nuclear protein that functions downstream of Sevenless to target a transcriptional repressor for proteolysis. In these cases, the genetic interactions may also be an indirect consequence of cells that are receiving an inductive signal remaining longer in a particular phase of the cycle than they would normally. The results presented here underscore the connection between the cell cycle and signaling pathways that govern cell determination. A less likely possibility is that the Cdc37, Peanut, and/or the 60-kD subunit of DNA primase are bifunctional proteins, with distinct functions in the cell cycle and in signal transduction (Chen, 2000b).
The covalent attachment of ubiquitin to cellular proteins is a powerful mechanism for controlling protein activity and localization. Ubiquitination is a reversible modification promoted by ubiquitin ligases and antagonized by deubiquitinating proteases. Ubiquitin-dependent mechanisms regulate many important processes including cell-cycle progression, apoptosis and transcriptional regulation. Ubiquitin-dependent mechanisms regulate synaptic development at the Drosophila neuromuscular junction (NMJ). Neuronal overexpression of the deubiquitinating protease Fat facets leads to a profound disruption of synaptic growth control; there is a large increase in the number of synaptic boutons, an elaboration of the synaptic branching pattern, and a disruption of synaptic function. Antagonizing the ubiquitination pathway in neurons by expression of the yeast deubiquitinating protease UBP2 also produces synaptic overgrowth and dysfunction. Genetic interactions between fat facets and highwire, a negative regulator of synaptic growth that has structural homology to a family of ubiquitin ligases, suggest that synaptic development may be controlled by the balance between positive and negative regulators of ubiquitination (Diantonio, 2001).
Synaptic morphology is dynamic; once formed, synapses expand, retract, and remodel throughout life. This plasticity underlies the refinement of neuronal circuits during development and may be critical for plasticity in the adult brain. To identify molecular mechanisms regulating the morphological growth of synapses, a genetic screen was performed for molecules whose neuronal overexpression disrupts synaptic growth control at the Drosophila NMJ. A collection of flies capable of the targeted overexpression of endogenous Drosophila genes was screened and two lines, EP(3)381 and EP(3)3520, were identified whose overexpression in the nervous system leads to synaptic overgrowth. Both EP(3)381 and EP(3)3520 overexpress fat facets (faf), a deubiquitinating protease. Endogenous faf transcript is strongly and widely expressed in the developing central nervous system (CNS), demonstrating that neuronal expression of faf from the EP lines produces overexpression, not misexpression, of the transcript (Diantonio, 2001).
Anatomical analysis at the NMJ reveals that neuronal expression from both EP(3)381 and EP(3)3520 leads to an increase both in the number of synaptic boutons and in the synaptic span (the extent of the muscle covered by the synapse). This increase is not seen in flies that do not overexpress faf v or that overexpress a non-functional faf gene (elav-Gal4 crossed to EP(3)381faf-). Neuronal overexpression of faf also causes an increase in the number of synaptic branches as quantified by the number of branch points. Postsynaptic expression of faf does not affect synaptic morphology (Diantonio, 2001).
To assess the physiological consequence of neuronal faf overexpression, both spontaneous and evoked neurotransmitter release were analysed at muscle 6 of third instar larvae. Despite the greatly expanded size of the NMJ with faf overexpression, the amplitude of evoked excitatory junctional potentials (EJPs) is markedly reduced. Given that the amplitude of miniature EJPs (mEJPs) shows only a small, albeit significant, reduction, a large decrease was measured in quantal content (the number of vesicles released by the nerve) as measured by dividing the EJP amplitude by the mEJP amplitude. Neuronal overexpression of faf also leads to a reduction in the frequency of spontaneous mEJPs. The reduction in both quantal content and mEJP frequency indicates a presynaptic defect in neurotransmitter release. Other presynaptic mutants with even greater reductions in quantal content do not show a structural overgrowth, thus the anatomical phenotype described above is probably a direct consequence of faf overexpression and not a secondary consequence of this physiological phenotype (Diantonio, 2001).
faf antagonizes ubiquitin-dependent mechanisms by deubiquitinating target proteins. Alterations in synaptic structure and function owing to overexpression of faf suggest that ubiquitin-dependent mechanisms normally act to regulate the developing synapse. However, faf, a characterized deubiquitinating protease, might have other functions. To investigate the role of deubiquitination in the regulation of synaptic development, transgenic flies were generated capable of the targeted overexpression of the yeast deubiquitinating protease UBP2. This enzyme antagonizes ubiquitin-dependent mechanisms in yeast and has overlapping substrate specificity with FAF+9. Overexpression of yeast UBP2 in the nervous system of Drosophila leads to marked synaptic overgrowth and a severe reduction in presynaptic transmitter release. This phenotype is very similar to that seen with faf overexpression. Hence, antagonizing ubiquitin-dependent mechanisms by overexpression of deubiquitinating proteases markedly affects synaptic development (Diantonio, 2001).
To identify molecular pathways regulated by faf, a genetic interaction screen was performed to identify genes that enhance the faf overexpression phenotype. The X chromosome was screened for viable mutations that are lethal in combination with neuronal overexpession of faf. 7,000 chromosomes were screened and 15 lethal enhancers, 12 of which form one complementation group, were identifed. These 12 mutants are alleles of the highwire (hiw) gene and share the synaptic overgrowth phenotype described for loss-of-function hiw mutants. The hiw loss-of-function phenotypes are very similar to the faf gain-of-function phenotypes described here, with a large increase in the number of synaptic boutons, branches, and synaptic span, a small decrease in quantal size, and a large decrease in quantal content. The hiw transcript encodes a greater than 5,000 amino-acid protein that is localized to synapses and that contains a RING-H2 finger, a domain recently identified in a large family of E3 ubiquitin ligases. Hence, a potential synaptic E3 ligase was identified as a lethal enhancer of neuronal overexpression of faf. This genetic interaction provides further evidence that ubiquitination may have a central role in regulating synaptic growth and function (Diantonio, 2001).
To further investigate the genetic relationship between hiw and faf, double mutants were generated between loss-of-function alleles of hiw and faf. faf loss-of-function mutants have phenotypes in the developing eye and female germ line. In faf mutants, no defects were found in either synaptic morphology or function, possibly due to genetic redundancy between faf and one of the 17 other putative deubiquitinating proteases in the Drosophila genome. Although no phenotype was found for faf mutants in otherwise wild-type flies, in the sensitized background of a hiw mutant a requirement for faf is found. Two different loss-of-function alleles of faf both suppress the physiological phenotype of hiw, leading to a more than doubling of both quantal content and mEJP frequency. Hence endogenous faf activity acts to inhibit neurotransmitter release in a hiw background, much as increased faf activity inhibits neurotransmitter release in an otherwise wild-type background. Mutants of faf do not suppress the synaptic overgrowth seen in hiw, indicating that the physiological and morphological phenotypes in hiw are genetically separable. This suggests that either these phenotypes are mediated by different hiw substrates, or the anatomical phenotype is more sensitive to disruption of ubiquitin-dependent mechanisms. Finally, the inability to suppress the small quantal size defect suggests that this phenotype, seen in both hiw mutants and with overexpression of faf, may be a secondary consequence of synaptic overgrowth (Diantonio, 2001).
Decreases in postsynaptic activity induce a compensatory increase in presynaptic transmitter release, demonstrating that a homeostatic mechanism regulates synaptic strength during development. To assess the relationship between homeostatic and ubiquitin-dependent regulation, glutamate receptor function was disrupted in a hiw mutant. Postsynaptic expression of a dominant negative glutamate receptor (DgluRIIA-M/R) in a hiw mutant leads to an 18% decrease in quantal size. Since homeostatic compensation still occurs in a hiw mutant, it is suggested that homeostatic and ubiquitin-dependent regulation are mechanistically distinct (Diantonio, 2001).
The data presented here indicate that ubiquitin-dependent mechanisms regulate synaptic development at the Drosophila NMJ and suggest that a balance between positive and negative regulators of ubiquitination controls the structure and function of the synapse. Antagonizing ubiquitination by the neuronal overexpression of the deubiquitinating proteases faf or yeast UBP2 leads to synaptic overgrowth and defects in neurotransmitter release. This phenotype is very similar to the loss-of-function phenotype of hiw, a putative synaptic E3 ubiquitin ligase. Gain-of-function mutants of faf enhance hiw and loss-of-function alleles of faf suppress hiw. It is proposed that hiw-dependent ubiquitination controls the level or activity of critical regulatory molecules at the synapse, and that these molecules can be deubiquitinated by faf and other deubiquitinating proteases. Ubiquitinated proteins have been identified at mammalian synapses, and ubiquitin-processing enzymes can regulate long-term potentiation and facilitation, therefore control of ubiquitination by molecules such as HIW and FAF could be a widely used mechanism for regulating synaptic growth and function (Diantonio, 2001).
Endocytosis modulates the Notch signaling pathway in both the signaling and receiving cells. One recent hypothesis is that endocytosis of the ligand Delta by the signaling cells is essential for Notch activation in the receiving cells. Evidence is presented in strong support of this model. In the developing Drosophila eye Fat facets (Faf), a deubiquitinating enzyme, and its substrate Liquid facets (Lqf), an endocytic epsin, promote Delta internalization and Delta signaling in the signaling cells. While Lqf is necessary for three different Notch/Delta signaling events at the morphogenetic furrow, Faf is essential only for one: Delta signaling by photoreceptor precluster cells, which prevents recruitment of ectopic neurons. In addition, the ubiquitin-ligase Neuralized (Neur), which ubiquitinates Delta, is shown to function in the signaling cells with Faf and Lqf. The results presented bolster one model for Neur function in which Neur enhances Delta signaling by stimulating Delta internalization in the signaling cells. It is proposed that Faf plays a role similar to that of Neur in the Delta signaling cells. By deubiquitinating Lqf, which enhances the efficiency of Delta internalization, Faf stimulates Delta signaling (Overstreet, 2004).
Cells with decreased lqf+ activity accumulate Delta on
apical membranes and fail to signal to neighboring cells. Three
Notch/Delta signaling events were examined in the eye: proneural enhancement, lateral
inhibition and R-cell restriction. Loss of lqf+-dependent
endocytosis during all three events has identical consequences to loss of
Delta function in the signaling cells. It is concluded that
lqf+-dependent endocytosis of Delta is required for
signaling, supporting the notion that endocytosis in the signaling cells
activates Notch in the receiving cells. However, Lqf is not required absolutely for all Delta internalization in the eye. Even in lqf-null cells, which are incapable of Delta signaling, some vesicular Delta is present. Perhaps not all of the vesicular Delta present in wild-type discs results from signaling (Overstreet, 2004).
Genetic studies in Drosophila indicate clearly that
deubiquitination of Lqf by Faf activates Lqf activity.
Moreover, genetic and biochemical evidence in Drosophila suggests
that Faf prevents proteasomal degradation of Lqf. In
vertebrates, however, it is thought that epsin is mono-ubiquitinated to
modulate its activity rather than poly-ubiquitinated to target it for
degradation. If Lqf regulation by ubiquitin also occurs this way in the
Drosophila eye, the removal of mono-ubiquitin from Lqf by Faf would
activate Lqf activity (Overstreet, 2004).
Whatever the precise mechanism, given that both Faf and Lqf are expressed
ubiquitously in the eye, two related questions arise. First, why is Lqf
ubiquitinated at all if Faf simply deubiquitinates it everywhere? One
possibility is that Faf is one of many deubiquitinating enzymes that regulate
Lqf, and expression of the others is restricted spatially. This could also
explain why Faf is required only for R-cell restriction. Another
possibility is that Faf activity is itself regulated in a spatial-specific
manner in the eye disc. Alternatively, Lqf ubiquitination may be so efficient
that Faf is needed to provide a pool of non-ubiquitinated, active Lqf.
Similarly, Faf could be part of a subtle mechanism for timing Lqf activation.
Second, why is Faf essential only for R-cell restriction? One possibility is
that there is a graded requirement for Lqf in the eye disc, such that
proneural enhancement requires the least Lqf, lateral inhibition somewhat more
and neural inhibitory signaling by R2/3/4/5 the most. The mutant phenotype of
homozygotes for the weak allele lqfFDD9 supports this
idea, as R-cell restriction is most severely affected. Alternatively, Lqf may
be expressed or ubiquitinated with dissimilar efficiencies in different
regions of the eye disc. More experiments are needed to understand the precise
mechanism by which the Faf/Lqf interaction enhances Delta signaling (Overstreet, 2004).
In neur mutants, Delta accumulates on the membranes of signaling
cells and Notch activation in neighboring cells is reduced. These results
support a role for Neur in endocytosis of Delta in the signaling cells to
achieve Notch activation in the neighboring receiving cells, rather than in
downregulation of Delta in the receiving cells. Because neur shows
strong genetic interactions with lqf and both function in R-cells,
Neur and Lqf might work together to stimulate Delta endocytosis. Lqf has
ubiquitin interaction motifs (UIMs) that bind ubiquitin. One
explanation for how Neur and Faf/Lqf could function together is that Lqf
facilitates Delta endocytosis by binding to Delta after its ubiquitination by
Neur. This is anattractive model that will stimulate further experiments (Overstreet, 2004).
One exciting observation is that the endocytic adapter Lqf may be essential
specifically for Delta internalization. Although, hedgehog, decapentaplegic and
wingless signaling pathways have not been examined directly, they appear to be functioning in the absence of Lqf.
These three signaling pathways regulate movement of the morphogenetic furrow and
are thought to require endocytosis. The furrow moves through lqf-null clones
and at the same pace as the surrounding wild-type cells.
Moreover, all mutant phenotypes of lqf-null clones can be accounted
for by loss of Delta function. Further experiments will clarify
whether this apparent specificity means that Lqf functions only in
internalization of Delta, or if the process of Delta endocytosis is
particularly sensitive to the levels of Lqf (Overstreet, 2004).
Lqf expands the small repertoire of endocytic proteins that are known
targets for regulation of cell signaling. In addition to Lqf, the endocytic
proteins Numb and Eps15 (EGFR phosphorylated
substrate 15) are objects of regulation. In vertebrates,
asymmetrical distribution into daughter cells of the alpha-adaptin binding
protein Numb may be achieved through ubiquitination of Numb by the
ubiquitin-ligase LNX (Ligand of Numb-protein X) and subsequent Numb
degradation. In addition, in vertebrate cells, Eps15 is phosphorylated
and recruited to the membrane in response to EGFR activation and is required
for ligand-induced EGFR internalization.
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are likely to provide an abundance of targets for its regulation (Overstreet, 2004).
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fat facets:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| Effects of Mutation
date revised: 15 March 2002
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