We have investigated the functions of the atlastin family of GTPases, emphasizing studies of atlastin-2 and -3. We had previously reported that the SPG3A
protein atlastin-1 co-localizes prominently with p115, a protein present at VTCs and cis
), mostly in brain, and also that atlastin-1 is present in growth cones and required for axon formation and elongation during development (13
). Interestingly, atlastin-2 and -3 are present widely in peripheral tissues and localize predominantly to ER membranes. Our findings suggest that, while all atlastins are present at the ER, VTCs, and cis
-Golgi cisternae to some degree, there are differences in distributions of the majority of each particular isoform that may correlate with some functional specificity of each atlastin along the ER-to-Golgi axis. Indeed, although both atlastin-2 and -3 localize to ER, we noted a difference in distribution between these two in that the atlastin-2 distribution on ER appeared more closely aligned with microtubules. Also, atlastin-1 localizes predominantly with VTCs/cis
-Golgi as well as within growth cones (12
), where dynamic ER has the ability to rapidly move into or away from the distal parts (24
). Clearly, however, there may be functional overlap among the highly homologous atlastins. Consistent with this notion, species such as Drosophila
have only one atlastin protein.
Because our experiments were carried out in HeLa cells, and levels of atlastin-1 are extremely low in these cells, we focused on knocking down atlastin-2 and -3 to assess for any intracellular changes. With the specific depletion of either atlastin-2 or -3 we saw two different types of changes in Golgi morphology, but no consistent ER phenotype. In cells where atlastin-2 and -3 double knockdowns were performed, we saw a similar degree of Golgi disruption, but also saw a more tubular ER morphology in a small subset of cells (unpublished data). In complementary experiments, we examined the structural and functional changes that occurred upon overexpression of either wild-type, SPG3A
-type mutant (20
), or dominant-negative forms of all three atlastins. In all cases, overexpression of the mutant, but not wild-type, atlastin proteins resulted in a change in ER morphology to a more tubular phenotype with very few interconnections by three-way junctions, which are points of homotypic fusion events in smooth ER that result in a polygon-like structure (26
). The fact that all six atlastin mutants defective in GTPase activity produced the same morphological change, while the wild-type atlastins did not, may reflect the fact that each overexpressed mutant atlastin protein can oligomerize with all three endogenous atlastins (Supplementary Material, Fig. S2
), thus impairing GTPase activity of heteromeric complexes comprising all of the atlastins. In assessing Golgi morphology, we found that overexpression of both the wild-type and SPG3A
-type constructs caused fragmentation of the Golgi into what appeared to be ‘mini-stacks,’ but this phenotype varied among the mutant forms. These differences may reflect functional specificity of the different protein or variations in expression levels within the cells. Expression of dominant-negative atlastin proteins produced a more consistent effect on Golgi morphology. However, on examining effects on protein trafficking in these overexpression paradigms, we found that there did not seem to be inhibition of VSVG trafficking to the plasma membrane, even in cells with significantly altered ER morphology.
One explanation for the differences between dominant-negative studies and siRNA depletion studies, particularly with respect to changes in ER morphology, is that the loss of a single isoform may have more modest effects because the other isoforms can compensate. Furthermore, even in the atlastin-2 and -3 double knockdown cells the degree of knockdown of each isoform may not be sufficient in a majority of the cells to elicit a phenotype, and there is also a low level of atlastin-1 in HeLa cells. On the other hand, the changes in Golgi morphology upon knockdown of a single atlastin may reflect more specialized functions of each isoform at the level of VTCs or Golgi apparatus. This notion is consistent with the fact that although both Golgi phenotypes were present upon knockdown of atlastin-2 or -3, there was a clear difference in proportion of cells displaying each phenotype in each atlastin siRNA condition. Thus, the majority of atlastin-2 siRNA cells exhibited more elongated Golgi tubules, whereas the majority of atlastin-3 siRNA cells had a more fragmented ‘mini-stack’ Golgi morphology.
We considered the possibility that, in our siRNA treatment conditions, the fragmented GM130- and YFP-Golgi-positive membranes may reflect Golgi proteins retained at ER exit sites, and consequently that the more elongated, tubular structures might represent Golgi proteins trapped within the ER. However, these GM130- and YFP-Golgi-labeled compartments were BFA sensitive, clearly demonstrating that they are formed after proteins have trafficked from the ER in the form of VTCs. It is likely that once VTCs have formed from the ER, atlastins are important in the movement of these vesicles along microtubules towards the MTOC (microtubule organizing center), the fusion of vesicular tubular complex (VTC) membranes, or perhaps both. The fact that these structures are BFA-sensitive is also consistent with our results showing that all atlastin siRNA cells examined were still able to traffic VSVG to the plasma membrane, with only a very small subset of cells showing a delay in trafficking. Indeed, secretory protein trafficking does not require a stacked Golgi morphology, nor does there need to be a centralized location of the Golgi apparatus (29
). This trafficking, however, may not be as efficient as in control cells, potentially accounting for the delay in VSVG trafficking seen in a small subset of atlastin-2 and -3 siRNA cells.
Thus, our results suggest that while depletion of atlastins by siRNAs and overexpression of dominant-negative, GTPase-deficient atlastin proteins have substantial effects on the morphology of the Golgi apparatus and the reticular ER structure, respectively, protein trafficking in the secretory pathway does not seem to be greatly affected. This is particularly interesting in light of a recent study that found that mutations in proteins critical for ER-to-Golgi transport in the secretory pathway affected development of dendrites far more than axons in Drosophila
). Conversely, since SPG3A is a long axonopathy, and loss of atlastin-1 causes effects predominantly on the development of axons in cultured cortical neurons (13
), atlastin-1, and possibly atlastin-2 and -3, may be important in other intracellular trafficking pathways, perhaps those involved in the establishment and maintenance of specific polarized membranes or membrane processes. Indeed, Sannerud et al
) have provided evidence for such a ‘Golgi-bypass’ pathway defined by Rab1 that is linked to the dynamics of the smooth ER as well as pre-Golgi intermediate compartments and functions in the delivery of membranes to developing neurites.
Mechanistically, the atlastin GTPases may serve an important role in fusion of ER membranes to form the more widely-spread ER reticulum by way of three-way junctions or else by influencing the association of ER with microtubules. Alternatively, atlastins may also play a role in VTC or Golgi ‘mini-stack’ movement along microtubules towards the MTOC as well as the formation of the normal stacked Golgi structure. In this regard, the interaction of atlastin-1, but not atlastin-2 or -3, with the microtubule-severing AAA ATPase spastin that is mutated in the most common HSP, SPG4, is particularly intriguing. It will be interesting to determine whether atlastin-2 and -3 interact with other microtubule-severing proteins, or whether this interaction with microtubule-severing enzymes is specific to atlastin-1 and represents an important functional specialization with relevance for the pathogenesis of the HSPs.
Importantly, spastin is also present at the ER, and this distribution is increased upon overexpression of atlastin-1. Furthermore, overexpression of SPG4
mutant spastin disrupts ER morphology in conjunction with microtubule abnormalities (21
), and overexpression of wild-type spastin results in increased lateral mobility of the translocon complexes that are part of membrane-bound polysomes (32
). Thus, proper ER morphology may be an important determinant of axon development and maintenance. Lastly, at least one additional HSP protein, the SPG17
protein seipin, localizes prominently to ER membranes (33
). Thus, in addition to proteins implicated in endocytosis that are mutated in a number of HSPs (4
), regulation of ER and Golgi morphology and possibly novel trafficking pathways important for membrane addition or dynamics at axons may also be a major theme in HSP disease pathogenesis.