Newly synthesized proteins and lipids of the secretory pathway are packaged into membrane carriers that bud from the endoplasmic reticulum (ER). These membranes fuse with each other and/or preexisting ER-Golgi intermediate compartment membranes (ERGIC) and are ultimately transported along microtubules toward the centrosome by the dynein motor protein complex. There are two noteworthy models regarding the next step. By the cisternal progression model, the membranes generate new cis-Golgi cisternae as they near the centrosome because of fusion with recycling vesicles bearing cis-Golgi components including processing enzymes. By the stable compartments model, the membranes fuse with preexisting cis-Golgi cisternae thereby delivering their content for processing. By either model, the continuous inward movement of membrane carriers will contribute to the steady state localization of Golgi membranes near the centrosome principally because the membranes bear active dynein at the time of their delivery. In addition, dynein is likely recruited directly from the cytoplasm onto Golgi membranes further contributing to Golgi pericentrosomal positioning. This section covers mechanistic details of dynein-based Golgi membrane movement (1.1) and also supporting roles in Golgi positioning of: anchoring connections between Golgi membranes and centrosomes (1.2), Golgi-nucleated microtubules (1.3), and bidirectional motility (1.4).
Moving in: Membrane Capture by Motors and Loading onto Microtubules
Motor proteins use the cytoskeleton network as highways for all membrane transport activities. Centripetal membrane movement is driven by forces generated mainly by the minus-end directed motor protein cytoplasmic dynein, a member of the Dynein superfamily (Schroer et al. 1989
; Kardon and Vale 2009
). Dynein moves along microtubules carrying bound cargo, such as Golgi membranes, using conformational changes driven by a cycle of ATP binding, hydrolysis and release. Dynein is a multimeric protein complex composed of catalytic heavy chains and noncatalytic intermediate, intermediate light, and light chains (A). Dynein heavy chain (DHC) exists in three isoforms. One of these isoforms, DHC1, assembles to form the dynein-1 motor complex, which is the major mediator of microtubule dependent minus-end directed movement in mammalian cells (Vaisberg et al. 1996
). Inhibition of dynein-1 causes the Golgi apparatus to fragment into stacks that are dispersed and immotile, suggesting that continuous dynein driven membrane transport is essential for centrosomal localization of the Golgi apparatus. DHC1 localizes both to Golgi membranes and those of the intermediate compartment (Roghi and Allan 1999
) and cultured cells from DHC1 knockout mice have a fragmented Golgi apparatus (Harada et al. 1998
). This is further strengthened by RNAi studies in which knockdown of components of the dynein-1 motor cause loss of Golgi positioning (Palmer et al. 2009
). There is conflicting data about the role in Golgi positioning of dynein-2, which contains the DHC2 isoform. DHC2 is localized primarily to Golgi membranes and its inhibition by isoform specific antibodies causes loss of Golgi positioning (Vaisberg et al. 1996
). However, siRNA mediated depletion of DHC2 fails to show any Golgi phenotype or loss of ER-Golgi transport (Palmer et al. 2009
). Indeed, other studies show a role for DHC2 in primary cilia biogenesis (Pazour et al. 1999
). Although dynein-1 is the primary motor implicated in Golgi positioning, a minus-end directed member of the kinesin motor family, KIFC-3, can also contribute, at least under the condition of cholesterol depletion. Adrenocortical cells cultured from Kifc3-/- mice show a fragmented Golgi and absence of inward Golgi motility but only after cholesterol depletion (Xu et al. 2002
). Whether dynein somehow depends on cholesterol for its activity remains to be tested.
Figure 1. The mammalian pericentrosomal Golgi ribbon. Fluorescent micrograph of cultured HeLa cells stained using antibodies against tubulin (green) and the Golgi marker protein giantin (red) shows relative positions of extensive microtubule network and the Golgi (more ...)
Figure 2. Dynein and dynactin schematic diagrams. The dynein motor protein complex is assembled on two catalytic heavy chains each containing a microtubule-binding domain, a motor domain consisting of six AAA ATPase modules, and a coiled-coil linker region (A). (more ...)
Each dynein molecule contains dimerized heavy chains. The carboxyl terminal region of each heavy chain comprises the motor domain and contains six triple-A ATPase domains and a microtubule binding stalk region. The amino terminal region acts as a scaffold that, in the dimer, binds two dynein intermediate chains (DIC) and two light intermediate chains (DLIC) (King et al. 2002
). Light chains LC-7 (roadblock), LC-8, and Tctex-1 can form homo- or hetero-dimers and assemble directly on DIC (Nikulina et al. 2004
; Kardon and Vale 2009
). Although the dynein heavy chain containing the ATPase domains at its carboxyl terminus is sufficient for imparting motility in in-vitro conditions, the assembly of the noncatalytic subunits at the amino terminus is required to mediate specific cargo-adaptor-motor linkages that couple motor movement to cargo movement on microtubules. For example, Tctex-1 binds the integral membrane protein rhodopsin to mediate transport of rhodopsin-bearing vesicles in photoreceptor cells (Tai et al. 1999
). Tctex-1 is also important for Golgi positioning because siRNA depletion of Tctex-1 blocks ER-Golgi traffic and fragments the Golgi apparatus. Indeed, all of the core subunits appear to comprise a functional unit for Golgi positioning as individual suppression by siRNA of DHC1, DIC-2, LIC1, Tctex-1, Roadblock, and LC-8 blocks ER-to-Golgi traffic and fragments the Golgi (Palmer et al. 2009
DIC not only acts as a scaffold for assembly of the light chain subunits of dynein but it also links the motor protein to dynactin a large regulatory complex (Vaughan and Vallee 1995
). Remarkably, dynactin consists of 11 different polypeptides that assemble forming two morphologically distinct domains: the actin related protein 1 (Arp1) rod and the p150 projecting-arm (B). In actin-like fashion, Arp1 assembles into a 40 nm filament and this associates with six other subunits forming the central rod-shape of dynactin. Near one end of the Arp1 rod, the carboxyl terminus of the elongated p150 subunit binds forming the projecting-arm onto which, at its amino terminus, are bound two other subunits, p50 (dynamitin) and p24 (Schroer 2004
). The Arp1 rod is thought to mediate interaction of dynactin with membranes because Arp1 is known to interact with β3-spectrin, which is a peripheral membrane protein (Holleran et al. 2001
). At its amino terminus, p150 has a microtubule-binding motif, the cytoskeleton-associated protein glycine-rich (Cap-Gly) motif (Waterman-Storer et al. 1995
). P150 also interacts with microtubule plus tip proteins EB-1 and Clip-170 (Askham et al. 2002
) and binds to DIC (King et al. 2002
). Therefore, via its Arp1 domain, dynactin binds membranes rich in spectrin and via the p150 arm; it connects the motor to microtubules. These interactions are sufficient for motility in vitro as spectrin-coated liposomes move on microtubules in the presence of the purified dynein-dynactin complex (Muresan et al. 2001
). Although it is clear that dynactin is involved in dynein-based motility there are several schools of thought regarding its exact role. These are: it confers dynein localization to microtubule plus tips (Vaughan et al. 2002
; Watson and Stephens 2006
), it activates dynein motor activity (King and Schroer 2000
), it tethers dynein to microtubules to promote motor processivity (Waterman-Storer et al. 1995
), it is an adaptor linking cargo to dynein (Holleran et al. 2001
; Muresan et al. 2001
), and it regulates bidirectional movement of membranes (Deacon et al. 2003
). Significantly, loss of dynactin from Golgi membranes in Arp1 mutant Drosophila
larvae or siRNA depleted cultured cells does not inhibit motor attachment to membranes but does inhibit both plus- and minus-end membrane motility (Haghnia et al. 2007
). Further investigation is required to clearly decipher the role dynactin plays in carrier motility and its bidirectional regulation.
RZZ (Rod-ZW10-Zwilch) is another complex that binds DIC and is implicated in Golgi positioning; however, its binding to DIC via ZW10 occurs primarily in mitosis for the purpose of spindle assembly, whereas its role in membrane motility probably involves interaction with the dynactin subunit dynamitin (Starr et al. 1998
). During interphase, ZW10 is ER associated (Hirose et al. 2004
) through the peripheral ER protein RINT-1 that binds the ER SNARE protein syntaxin-18 (Arasaki et al. 2006
). Interestingly, the ZW10 amino-terminal domain binds RINT-1 and dynamitin in a mutually exclusive manner (Inoue et al. 2008
). This could be the basis of cycling between ER and Golgi membranes. In any case, dominant negative, knockdown, and antibody inhibition experiments all show loss of Golgi positioning and decreased minus-end Golgi motility (Varma et al. 2006
Initiation of inward movement of ER-derived transport carriers by dynein can occur through direct recruitment of dynein from the cytosol (A), through binding of dynein preloaded on plus-end tips of microtubules to membranes (B), or through delivery of dynein via recycling vesicles (C). These modes are not necessarily exclusive. Blocking preloading by knockdown of EB1, which is required for dynactin association with the plus tips of microtubules, has no apparent effect on ER to Golgi motility suggesting that although preloading takes place it is not required (Watson and Stephens 2006
). Recycling of dynein from the Golgi to the ERGIC on membranes has not been observed but it is an interesting possibility suggested by the general finding that membranes show bidirectional motility. In contrast to the other modes, recycling would place a premium on regulating motor activity as opposed to motor recruitment.
Figure 3. Inward membrane movement. A pericentrosomal Golgi ribbon network is depicted (A). Minus-ends of microtubules converge at the centrosome and support inward movement by dynein of secretory and Golgi membranes derived from the ER. Also shown are three modes (more ...)
In principle, recruitment on membranes can occur by direct binding of the motor to membrane lipids, binding of the motor to peripheral components that bind lipids, or binding to a compartment-specific protein receptor. Direct membrane binding is observed in protease-treated synaptic membranes (Lacey and Haimo 1994
). Synthetic acidic phospholipid vesicles bind dynein and binding increases dynein ATPase activity (Ferro and Collins 1995
), but the physiological relevance of direct lipid binding by the motor remains unclear. Changes in lipid composition seem unlikely to account for the specificity of dynein membrane interactions. Further, motility of these vesicles is poor and is markedly improved by cytosol addition suggesting the participation of additional factors (Muresan et al. 2001
). A key cytosolic component may be β-spectrin acting to bind lipids and recruit the motor (A). β-spectrin binds both acidic phospholipids and dynactin and is sufficient to substitute for cytosol in conferring full motility in vitro (Muresan et al. 2001
). However, β-spectrin is localized to many types of membranes again raising the question of how specificity of motor recruitment is obtained (De Matteis and Morrow 2000
). A more specifically localized candidate is the peripheral COPII vesicle coat complex component sec23, which interacts directly with the p150 subunit of dynactin (Watson et al. 2005
). The sec23/motor connection also has problems because the coat is only transiently present on the membrane and although disruption of dynactin binding to sec23 lowers kinetics of ER to Golgi transport, membrane transport velocities on microtubules are unaffected (Watson et al. 2005
; Fromme et al. 2008
Figure 4. Membrane recruitment of dynein. Golgi membranes are moved inward by dynein moving on microtubules and one possibility is that dynactin links dynein to Golgi membranes by binding spectrin (A). In light of dynactin-independent dynein membrane association (more ...)
The final category, a compartment specific receptor (B), is appealing but the current candidates each have limitations in explaining how dynein is membrane recruited for ER-to-Golgi transport and Golgi positioning. There are a few convincing cases of membrane proteins that bind dynein directly but these are unlikely to be generally involved in either ER-to-Golgi traffic or Golgi positioning. One is rhodopsin, for which the interaction serves to confer dynein-based motility of rhodopsin-containing vesicles, but rhodopsin is only expressed in photoreceptor cells (Tai et al. 1999
). Another is Drosophila
bicaudal-D and its mammalian homolog BICD2, which localizes to Golgi membranes, microtubule plus tips, and centrosomes (Hoogenraad et al. 2001
; Fumoto et al. 2006
). Via its amino terminus, BICD-2 binds dynein-dynactin and via its carboxyl terminus, it binds the GTPase rab6 thereby recruiting dynein to rab6 positive membranes (Matanis et al. 2002
). Induced targeting of BICD-2 to mitochondria or peroxisomes causes pericentrosomal clustering of the membranes and enhances recruitment of dynein on these membranes (Hoogenraad et al. 2003
). Nevertheless, BICD-2 localizes to the TGN, and is involved in COPI independent Golgi to ER transport (Matanis et al. 2002
). Further, BICD-2 knockdown does not alter Golgi positioning (Fumoto et al. 2006
), hence its involvement in ER-to-Golgi transport and Golgi positioning is unlikely. Golgin-160 and GMAP210 are much better candidates in terms of localization and phenotype. Each is a cis
-Golgi protein required for minus-end movement of Golgi membrane (Rios et al. 2004
; Yadav et al. 2009
) and GMAP210 targeted to mitochondria induces their clustering. Nevertheless, whether either protein participates in motor recruitment is unknown. Lava lamp is a peripheral Golgi-associated golgin required for cellularization in the Drosophila
embryo (Sisson et al. 2000
; Papoulas et al. 2005
). Lava lamp provides a link for the motor through associations with spectrin, the dynein complex and CLIP-190 and inhibition of lava lamp blocks dynein-dependent clustering of Golgi membranes. There are no studies of lava lamp outside Drosophila
and a mammalian homolog for lava lamp has not yet been identified.
As should be evident, the frequently cited candidates for receptor complexes mediating membrane association of dynein each have shortcomings. For example, dynactin knockdown leaves dynein on the Golgi, BICD-2 is not required for Golgi positioning and lava lamp and others appear to have restricted expression. Further, as noted previously (Linstedt 2004
), it is confounding that inhibition or depletion of a multitude of components perturbs Golgi positioning (). Presumably, this reflects both the dynamic nature of the Golgi apparatus and the dependence of its integrity on many pathways. Thus, the following straightforward criteria for a bona fide receptor complex can be proposed.
Select proteins showing Golgi phenotypes following knockdown.
- Knockdown or inhibition must block both dynein localization and Golgi motility.
- Localization must coincide with dynein on Golgi membranes.
- Two domains should be evident, one that directly binds a dynein component and one that mediates Golgi localization.
- Reconstitution in artificial membranes should confer dynein recruitment and motility.
- Because dynein dissociates from Golgi membranes during mitosis (see below), interaction of dynein with the receptor, or interaction of the receptor with the membrane should be regulated.