The eukaryotic Golgi complex is an essential organelle involved in many cellular processes, including lipid biosynthesis, protein modification and intracellular membrane trafficking. A major question and area of debate in cell biology is how the Golgi complex is inherited by daughter cells during mitosis. This has led to two different mechanistic models. One model proposes that the Golgi is a derivative of the endoplasmic reticulum (ER) and arises de novo from the ER during late telophase, whereas the other model proposes the Golgi complex is a unique organelle that arises by a template-based mechanism, requiring existing Golgi subunits for reassembly after mitosis (Eisman, 2006 and references therein).

This study examines the localization and function of a Golgi protein encoded by centrosomin's beautiful sister (cbs) during cleavage in Drosophila. Cbs contains a GRIP domain that is 57% identical to vertebrate Golgin-97. Cbs undergoes a dramatic relocalization during mitosis from the cytoplasm to an association with chromosomes from late prometaphase to early telophase, by a transport mechanism that requires the GRIP domain and Arl1, the product of the Arf72A locus. Additionally, Cbs remains independent of the endoplasmic reticulum throughout cleavage. The use of RNAi, Arf72A mutant analysis and ectopic expression of the GRIP domain, shows that cycling of Cbs during mitosis is required for the centrosome cycle. The normal cycling of Cbs is important for normal centrosome maturation, replication and spindle attachment during mitosis, and the linkage of this process to the normal nuclear cycle The effects on the centrosome cycle depend on Cbs concentration and Cbs transport from the cytoplasm to DNA. When Cbs levels are reduced centrosomes fail to mature, and when Cbs transport is impeded by ectopic expression of the GRIP domain, centrosomes undergo hypertrophy. It is proposed that, Cbs is a trans-Golgi protein that links Golgi inheritance to the cell cycle and the Drosophila Golgi is more vertebrate-like than previously recognized (Eisman, 2006).

In vertebrate cells, it is generally agreed that, during mitosis the Golgi breaks down into individual stacks at prophase, which then fragment into many mitotic Golgi vesicle clusters by prometaphase. Do clusters merge with the ER at mitosis and re-emerge at telophase to self-assemble the Golgi, or do they remain intact and independent of the ER during mitosis, acting as a template to reform the Golgi after mitosis? Persuasive evidence exists that mitotic clusters remain independent of the ER throughout mitosis, and that clusters reorganize during metaphase to associate with the mitotic spindle, centrosomes and metaphase plate during mitosis (Jesch, 2001; Jokitalo, 2001; Shima, 1998). It has also been shown that, if the Golgi is removed from cytoplasts, the Golgi complex does not form de novo from the ER. Additionally, the initiation of Golgi fragmentation and dispersal, prior to cluster formation, is required for cells to enter mitosis and may act as a Golgi-damage sensor regulating the cell cycle, which would serve to link the Golgi cycle to the centrosome cycle (Eisman, 2006 and references therein).

In Drosophila, the morphology and mitotic behavior of the Golgi is variable, based on cis-Golgi proteins and depending on the tissue type investigated. During syncytial embryogenesis maternally supplied Golgi membranes are concentrated at the embryonic cortex as several thousand punctate structures that remain unchanged during the syncytial nuclear divisions. The ER is present at the embryonic cortex and as an interconnected membrane network throughout the cytoplasm of the oocyte at fertilization. Additionally, the cortical ER and Golgi are present as a continuous network in syncytial preblastoderm embryos; this network fragments into individual units associated with nuclei following nuclear migration to the cortex in syncytial blastoderm embryos (Frescas, 2006). In cellular embryos and in Drosophila tissue culture cells the interphase Golgi exists as discrete units throughout the cytoplasm that break down and disperse during mitosis. Finally, in imaginal disc cells during pupariation, the Golgi is present as multiple stacked cisternae, which form from pre-existing Golgi units; and these cisternae have polarity (Yano, 2005), suggesting that the Drosophila Golgi has distinct functional compartments (Eisman, 2006).

Several peripheral membrane proteins called golgins have been identified that are involved in formation of the Golgi matrix and that link the Golgi to the cytoskeleton. These proteins function within specific domains of the Golgi, have a coiled-coil structure, and are regulated by small GTPases (Barr, 2003). Several cis-Golgi matrix golgins can reform a structural Golgi skeleton, in the absence of ER-derived Golgi membranes, suggesting that the matrix represents a template that gives the Golgi an autonomous identity (Seemann, 2000). Mitotic Golgi-vesicle clusters are enriched for matrix golgins and these vesicles maintain a cis to trans polarity throughout mitosis (Shima, 1997). This has led to the proposal that these vesicles are required for inheritance of the Golgi during mitosis (Seemann, 2002). In vertebrates, the golgins are clearly important for normal Golgi structure and regulation of the cell cycle. In Drosophila several cis-Golgi golgins have been investigated, but very little is known about the trans Golgi and the proteins that are specific to this compartment (Eisman, 2006).

A subset of peripheral membrane golgins contain a 42-amino-acid, functionally conserved Golgi-localization GRIP domain, which acts to localize vesicles to the trans Golgi (Barr, 1999; Kjer-Nielsen, 1999). Subsequent work has shown that this functional domain is highly conserved across the eukaryotes (Gilson, 2004; Luke, 2003; McConville, 2002). The function of GRIP proteins is still poorly understood, but ectopic expression of either the human Golgin-245 or Golgin-97 GRIP domains in HeLa cells leads to the displacement of endogenous GRIP proteins and mislocalization of other trans-Golgi proteins. This expression was also associated with a disruption of the trans-Golgi structure and interference with vesicle trafficking (Yoshino, 2003). The expression of either the Golgin-97 or Golgin-245 GRIP domains causes similar defects in Golgi structure, suggesting that the GRIP domains from both proteins share a common binding partner. However, both endogenous proteins form homodimers and thus potentially have independent functions (Luke, 2005). These findings make the GRIP domain a reliable marker of the trans Golgi in eukaryotes (Eisman, 2006).

Most GRIP proteins are regulated by the ADP-ribosylation-factor-like (ARL) GTPases (Barr, 2003). The GTP-bound form of Arl1 GTPase is required for recruitment of the GRIP domain to the trans Golgi in yeast (Behnia, 2004; Panic, 2003b) and vertebrates (Lu, 2003). The Arf72A gene, which encodes Arl1 in Drosophila has been identified and an embryonic recessive lethal allele has been recovered (Tamkun, 1991). Unfortunately, the mutant phenotype has not been investigated. Although Arl1 interacts with other, non-GRIP-containing proteins (Panic, 2003b), its interaction with most eukaryotic GRIP domains appears to be highly conserved (Eisman, 2006).

The Golgin-97 GRIP data discussed above suggests a function in maintaining Golgi structure, possibly as a component of the trans-Golgi matrix. Injection of anti-Golgin-97 antibody into cells causes the fragmentation of the entire Golgi complex, supporting a matrix function required for the global organization of this organelle (Lu, 2004). Interestingly, Golgin-97 also localizes to the centrosome throughout the cell cycle and it may act as a Golgi-nucleating protein at the centrosome, analogous to γ-tubulin nucleation of microtubules (Takatsuki, 2002). Golgin-97 is emerging as an important component of the trans Golgi in vertebrates, but has yet to be characterized in any other organism (Eisman, 2006).

This study reports the initial functional analysis of the maternal products of the Drosophila gene centrosomin's beautiful sister (cbs), during early embryogenesis. cbs encodes three isoforms, including a functional homolog of vertebrate Golgin-97. During mitosis Cbs protein undergoes a dramatic subcellular relocation, similar to the relocation described for the vertebrate Golgi matrix during mitosis. Cbs and the GRIP domain are required for maintenance of the Drosophila trans-Golgi complex during embryogenesis, and for normal centrosome maturation during the cell cycle. These results suggest that several characteristics of the Drosophila trans Golgi during the cell cycle are similar to vertebrates, making this a useful system for investigating the compartmentalization and inheritance of this complex organelle (Eisman, 2006).

The complex structure of the vertebrate Golgi, its pericentriolar location in cells and the mechanism of inheritance for this organelle appears to be a unique characteristic of vertebrates. The Drosophila Golgi complex exists as many discrete stacks during syncytial development and, on the basis of staining for the cis-Golgi proteins ßCop and p120, these stacks apparently remain unchanged during mitosis. Because of this, the embryonic Drosophila Golgi has been largely overlooked as a useful system to study this organelle during mitosis. However, this study of Cbs suggests that this is an oversight and that the inheritance and structure of the fly Golgi shares several features with the vertebrate organelle (Eisman, 2006).

This study presents the initial characterization of three golgin-like isoforms of Cbs that play a role in Golgi inheritance and the centrosome cycle in Drosophila during embryogenesis. It is proposed that two isoforms of Cbs are Golgin-97-like and, on the basis of the following, at least the GRIP-containing isoform functions at the trans Golgi. (1) The initial in silico prediction that all long isoforms of Cbs are golgin-like, suggested that Cbs is a structural protein of the relatively unknown trans-Golgi matrix in Drosophila. (2) One Cbs isoform contains a GRIP domain, which has been shown to be a functionally conserved trans-Golgi-targeting sequence in eukaryotes, making the GRIP domain a useful marker of the trans Golgi. (3) Cbs and the Drosophila Golgi protein Lva colocalize at chromosomes during congression and at the pericentriolar region during cellularization in Drosophila embryos. (4) In an Arf72A-mutant background, Cbs fails to localize to DNA during mitosis, leading to a significant reduction in Cbs and an apparent complete loss of Lva in early embryos. (5) GFP::GRIP fusion peptide is transported to the centrosomes and mitotic spindles during mitosis and interferes with the transport of endogenous Cbs protein during mitosis and normal centrosome function (Eisman, 2006).

The dramatic subcellular relocation of Cbs during mitosis at all stages of embryogenesis investigated, clearly correlates with the cell cycle and might reflect a unique mode of inheritance for a fraction of the Golgi. During embryogenesis the ER and Cbs remain independent of each other at all stages of mitosis, similar to vertebrate Golgi-matrix proteins, including Golgin-97 (Seemann, 2002; Takatsuki, 2002; Yoshimura, 2001). This supports an ER-independent mechanism for the inheritance of this fraction of the Golgi in Drosophila, whereas the association of the protein with the chromosomes argues for a chromatin-coupled mechanism for the inheritance of this Golgi component (Eisman, 2006).

Although many aspects of Cbs localization are similar to the reorganization of the animal Golgi during mitosis, the association with chromosomes from prometaphase to early telophase is unprecedented for any of the known Golgi proteins. In vertebrates, the Golgi ribbon breaks down sequentially, forming fragments around nuclei during early prophase and the mitosis entry requirement were both described by Sutterlin (Sutterlin, 2002), and the formation of mitotic vesicle clusters was described by Shima (Shima, 1998; Shima, 1997). During telophase these clusters are involved in the ATP-dependent, sequential reassembly of individual cisternae and the Golgi ribbon in vertebrates. If the embryonic Drosophila Golgi exists as individual Golgi units, the initial prophase changes in Cbs may be analogous to the initial Golgi fragmentation in vertebrates, providing a link between Golgi inheritance and the cell cycle in Drosophila. The microtubule-dependent reorganization of Cbs during late prophase and prometaphase would then correspond to the second phase of vertebrate Golgi fragmentation, when mitotic clusters associate with astral microtubules. As noted, the subsequent transport of Cbs to mitotic chromosomes may represent an efficient and reliable means of inheriting a fragmented organelle in a syncytial, rather than a cellular environment (Eisman, 2006).

The first potential interphase function of Cbs occurs during cellularization, when the approximately 6000 cortical nuclei cellularize simultaneously to form the cellular blastoderm. During cellularization, Cbs and Lva colocalize in the pericentriolar region, which is proposed to correspond to the site of the trans Golgi in Drosophila. In addition to Lva, projections from the ER colocalize with the pericentriolar Cbs region during cellularization and gastrulation, possibly for the direct transfer of new membrane from the ER to the trans Golgi. Taken together these data suggest that, at developmental stages that require large amounts of membrane production and transport, at least the trans Golgi may form a single, large continuous structure that maintains a pericentriolar location in cells and a close association with the ER, much like the vertebrate Golgi complex (Eisman, 2006).

RNAi depletion, Arf72A¹-mutant analysis and GFP::GRIP ectopic expression data provide insight into the function of Cbs and the GRIP domain during embryogenesis. The RNAi depletion of the maternal pool of cbs transcript causes a significant reduction in the protein levels of Cbs, Centrosomin (Cnn) and Lva during the syncytial cleavage divisions. Additionally, Cnn fails to properly localize to mitotic centrosomes. If the loss of these proteins is excessive development fails, but if some minimal level of Cbs is present development appears to proceed normally (Pelletier, 2000), showing that after removing the Golgi in cytoplasts, the addition of 2-5% of the normal amount of Golgi restored 35% of the transport function assayed (Eisman, 2006).

Unlike RNAi-depleted embryos, in Arf72A-mutant embryos, Cbs localization during syncytial prophase is similar to wild type, but transport of Cbs to the chromosomes is completely abrogated. When Cbs is not inherited by a chromosome-based mechanism during the syncytial blastoderm stage, Cbs and Lva protein accumulation is decreased similar to RNAi depletion of Cbs, and cellularization fails, suggesting that GRIP function is required for the inheritance and maintenance of the Golgi. In Arf72A¹-mutant embryos, the effect on the centrosome cycle during early syncytial blastoderm stages is less severe than RNAi but, by cellularization and early gastrulation, Cbs is nearly absent and the incidence of centrosomal defects increases, including centrosome fusions and loss of centrosomes. These data suggest that, GRIP domain function is required to maintain high levels of Cbs during late cleavage, so that normal prophase cycling of Cbs can occur. When Cbs levels are significantly reduced (similar to Cbs depletion by RNAi) Cbs fails to form aggregates during prophase and Cnn fails to localize to the centrosomes. The finding that this early relocation of Cbs might not require microtubules is similar to the finding that Cnn localization to the centrosome is by a microtubule-independent process (Eisman, 2006).

The ectopic expression of a GFP::GRIP fusion protein supports a role for this domain in the inheritance of Cbs via the chromosomes, and links Cbs to the centrosome cycle. In GFP::GRIP-expressing embryos, both the fusion protein and endogenous Cbs are transported to chromosomes but Cbs appears to bind to chromosomes more efficiently than the fusion protein. Nevertheless, large particles of Cbs accumulate in the cytoplasm during mitosis, similar to the Arf72A mutant phenotype. This block in Cbs transport also leads to an excessive accumulation of Cnn at centrosomes, causing centrosome hypertrophy and re-replication. These data suggest that, the transport of Cbs and Cnn from the cytoplasm to chromosomes and centrosomes, respectively, is linked in a time-dependent manner. The GRIP protein could interfere with Cbs transport by competing with Cbs for Arl1 binding, by affecting transport-machinery binding or a combination of the two. Alternatively, accumulation of the GRIP peptide at centrosomes might physically block transport of Cbs by directly interfering with Cnn function. Discerning between these possibilities will, of course, require further investigation. Nonetheless, it is clear that the normal cycling of Cbs is important for normal centrosome maturation, replication and spindle attachment during mitosis, and the linkage of this process to the normal nuclear cycle (Eisman, 2006).

Taken together, these results support a model that requires Cbs function for inheritance and maintenance of a subset of the Golgi complex in Drosophila, and links Golgi inheritance to the cell cycle via the centrosome cycle. During syncytial cleavage divisions, it is proposed that the initial prophase changes in Cbs are required for centrosome replication and maturation by an Arl1-independent mechanism, because both processes are normal in an Arf72A-mutant background, provided sufficient levels of cytoplasmic Cbs particles form during prophase. In RNAi-depleted animals the maternal load of cbs transcripts is reduced, resulting in an early loss of Cbs and an increase in centrosome defects. Because Cbs particles do not need to associate with centrosomes for normal replication and maturation, an indirect link is proposed between Cbs and Cnn cycles, requiring a set period of time of Cbs particle formation for sufficient Cnn localization to centrosomes. The combined data suggest that, when Cbs levels are reduced there is not sufficient time for localization of Cnn to centrosomes and, conversely, when the traffic time is extended by the ectopic expression of the GRIP domain, Cnn localization continues, resulting in centrosome hypertrophy. This mechanism functions only during prophase, because reduced centrosomes fail to mature during late mitosis in RNAi mutants and centrosome replication does not occur after prophase in embryos that express the GRIP fusion peptide (Eisman, 2006).

This characterization of Cbs suggests that many aspects of Golgi inheritance in animal cells are more similar between flies and vertebrates than previously perceived. Although the Drosophila Golgi complex is much less complex than that of the vertebrate, flies offer a powerful genetic and molecular in vivo animal system that represents an evolutionary intermediate, useful for addressing several questions regarding the animal Golgi that are difficult to address using tissue culture and in vitro systems alone. Even though many Golgi components in Drosophila remain uncharacterized, Cbs, Lva and several other cis-Golgi proteins offer a starting point for a more complete understanding of the role and regulation of golgins in Golgi structure and inheritance, how Golgi-vesicle transport is linked to the microtubule cytoskeleton and how the inheritance of this organelle is linked to the cell cycle in animals. Future work on Cbs and the Drosophila Golgi promises to be interesting and useful for reaching a more complete understanding of this fascinating organelle (Eisman, 2006).

GENE STRUCTURE

cDNA clone length - 2179bp (CG4840-RA)

Bases in 5' UTR - 161

Exons - 6

Bases in 3' UTR - 182

PROTEIN STRUCTURE

Amino Acids - 611 (isoform A)

Structural Domains

cbs (CG4840) was identified in studies of centrosomin (cnn). The cbs locus lies adjacent to cnn in the 50A region of the right arm of the second chromosome in Drosophila. An initial characterization included screening of cDNA libraries and expressed-sequence-tag (EST) collections to produce transcript profiles for both genes, and saturation mutagenesis screens to recover recessive lethal and sterile mutations. Multiple mutations were uncovered in cnn and other genes in this region, but no mutations were recovered cbs (Eisman, 2006).

The cbs gene is approximately 3 kb long and contains two transcription start sites located 134 and 278 base pairs distal to the most distal cnn transcription start site. The gene encodes at least five transcripts, which translate into a total of three different acidic protein isoforms. The two larger isoforms differ by the presence or absence of a functional GRIP domain and are similar to human Golgin-97. The longest isoform of Cbs is 611 amino acids long and has a predicted molecular mass of 70.8 kDa. Although the overall sequence conservation between Cbs and Golgin-97 is moderate this isoform contains a 42-amino-acid GRIP domain located near the C-terminus (aa 551-593) that is 57% identical to the GRIP domain in human Golgin-97. The alternatively spliced long Cbs isoform is missing amino acids 558 to 567, which eliminates the GRIP motif. The third short Cbs isoform is 367 amino acids long, has a predicted molecular mass of 42.7 kDa and is also similar to several golgins (Eisman, 2006).

The predicted secondary structure of Cbs is predominately coiled-coil, with short disordered regions that interrupt the coils, similar to the structure of all known golgins (Barr, 2003). There are also nine putative phosphorylation sites, consistent with the finding that several golgins undergo post-translational regulation (Dirac-Svejstrup, 2000; Preisinger, 2001) and three putative myristylation sites, necessary for post-translational modifications associated with protein-membrane interactions (Eisman, 2006).

On a western blot of 0-hours- to 2-hours-old embryos, a strong Cbs band was detected that runs just below the 77 kDa marker and a relatively weaker band that runs below the 50 kDa band of α-tubulin 84B, but well above the 34 kDa marker. The apparent molecular mass of both bands is in close agreement with the predicted sizes for the three Cbs isoforms, although both bands run slightly higher than expected. On the basis of western blot data and the isolation of both long isoforms from embryonic cDNA libraries, all three isoforms are present in early Drosophila embryos. Taken together these data suggest that Cbs produces three, potentially functionally distinct, golgin-like peripheral membrane proteins of the Drosophila trans Golgi (Eisman, 2006).

date revised: 5 March 2007

Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.