Intertwined And Finely Balanced: Endoplasmic Reticulum Morphology, Dynamics, Function, And Diseases Part 7

3.4. Computational Analysis of ER Dynamics

Analysing the dynamics of the ER in vivo is challenging due to its complex morphology and rapid dynamics as well as the phototoxicity of fluorescence microscopy experiments. Mathematical modeling of the network is an opportunity to gain further insights into this complex system from a different viewpoint. 

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ER tubules can be regarded as semiflexible polymers, with junctions represented by cross-links. Recently, many groups have modeled the dynamics of such cross-linked networks of semi-flexible polymers [263–266], including those influenced by active forces such as molecular motors [267–270]. Such models may provide insights into ER network dynamics by simplifying the system and only considering interactions with microtubules. 

A recent study modeling the motion of a particle surrounded by a network of semi-flexible polymers may provide insights into the functional reason for active rearrangements of the ER. Gong et al. [270] found that the diffusion of a particle embedded in a network of semi-flexible polymers was enhanced by the presence of active motors. 

Diffusion of particles in a network with active motors was super-diffusive, whereas, without active motors, sub-diffusion was observed. A possible explanation given for this enhanced motion was the greater number of collisions between the particle and the network in the presence of active motors. 

This result may relate to the dynamics of particles in the lumen of the ER. Fluctuations in the tubules and sheets of the network may induce faster dynamics of the particles within the lumen due to more frequent collisions with the lumenal face of the lipid bilayer. 

It has also been shown that certain aspects of plant ER morphology and dynamics can be replicated by considering the ER as a minimal network [271–273], where points are connected using the minimum length of tubules possible. This model accurately recreates aspects of ER morphology and dynamics. The structure of tubular regions of the network is accurately recreated, as are the dynamics of ring closure. 

This approach, however, is as yet unable to relate the rapid rearrangement dynamics of the network found using the model to those observed experimentally [273]. The motion of lumenal particles has also recently been simulated. 

A model describing the dynamics of particles within the lumen of the ER searching for reaction partners concluded that the morphological properties of the network are the most influential factors in determining the exploration times of molecules [274]. 

Future improvements in imaging data, as well as in the models used to represent the ER network, will result in improvements in both the conclusions of computational models and workflows for analyzing experimental data. 

Recently, several groups have released open-source software designed to extract dynamic variables from videos of the ER in live cells [24,27]. Such methods to quantify the morphology and dynamics of the ER network are essential to advance our understanding of this organelle. 

Analyzing the changes in dynamics and morphology under various cellular conditions will shed light on the underlying reason for ER dynamics and how altered dynamics may lead to clinical pathologies.

4. Morphology, Dynamics & Disease

It is unsurprising that an organelle such as the ER, with its critical and diverse functions, would be involved in many diseases. Dysregulation of the morphology and distribution of the functional subdomains of the ER (as described in Section 2.2) is therefore likely to affect their function and may result in disease states, particularly in neurons [32].

Many ER morphology-regulating proteins are known to be mutated in human diseases [227]. Miscommunication between the ER and mitochondria has also been identified as a contributor to many diseases, either via dysregulation of calcium ion transfer, insulin signaling, or mitochondrial division. 

Interactions between the ER and endosomes/lysosomes are similarly vital for normal cellular function, with particular problems being seen in neurons when proteins involved in these interactions are mutated [195,227]. 

Additionally, many organisms that give rise to pathogenic infections rely on ER machinery for their replication or entry into the cell. The best-characterised proteins involved in both diseases and ER maintenance are summarised in Table 1. 

Another major factor in many diseases, particularly cardiovascular diseases, is ER stress. MCSs are affected by many other types of stress [85,275], and so this is likely to prove an important factor in the disease. Further work is needed to fully explore the interplay between ER morphology and ER stress. Links between excessive ER stress, UPR activation, and the onset of diseases were recently reviewed [276–278], but are not addressed here.

ER morphology-regulating proteins are implicated in many diseases, including multiple neurodegenerative disorders (see [227] for an excellent, thorough review). Atlastins, a family of proteins that mediate the fusion of ER tubules to the network [4,348], are known to be mutated in hereditary spastic paraplegia (HSP) [285] and hereditary sensory and autonomic neuropathy (HSAN) [346,347]. 

REEPs and protrudin, which are known to stabilize regions of high membrane curvature in the ER [2,281], are also mutated in some forms of HSP [280,286], suggesting that abnormal ER morphology plays a role in the disease. ER-late endosome/lysosome MCSs are frequently seen in neurons [70] and are vitally important given that they are commonly affected by disease-causing mutations (Table 1; [227]). Protrudin mutations may affect ER distribution in axons, due to its interactions with kinesin-1, and its role in generating motile ER tubule-late endosome MCSs [205].
This function is vital for efficient neurite extension [129,195,204,227]. Mutations in KIF5A could conceivably affect this pathway. The disruption of ER–endosomal interactions also affects endosomal sorting and leads to lysosomal defects (Section 2.2.6 and 3.1.2), and this is frequently linked to diseases such as HSP and ALS (Table 1), where it can be caused by mutations in spastin, strumpelin, REEP1 [177], and VAPB, as described elsewhere in this special issue [291]. 

Mutations in proteins regulating ER morphology and dynamics have also been associated with amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Warburg Micro syndrome and spinocerebellar ataxia type 2. 

Reticulons 3 and 4 are mutated in Alzheimer's disease [336] and ALS [299], respectively. Reticulons regulate ER membrane curvature [2] and their mutation in ALS changes the distribution of chaperone proteins within the ER [299] and therefore is likely to negatively affect ER function. Recently, Mookherjee et al. discovered that protein aggregation in the cytoplasm, a common hallmark of neurodegenerative diseases, affects both ER morphology and dynamics [359]. 

Reticulon 4 was found to bind to the cytoplasmic aggregates, which may be the cause of the abnormal ER structure observed. Fewer three-way junctions, slower lumenal dynamics, and impaired tubule fusion efficiency were observed in cells with protein aggregates. 

Therefore not only do morphology-regulating proteins play a role in the onset of the disease, but they may also influence cellular processes once the disease has progressed. 

Warburg Micro syndrome, a rare disorder that results in neurodevelopmental defects, can be caused by mutations in Rab18 [29]. Rab18 is involved in several ER-related processes including the regulation of ER-lipid droplet MCS involving the NRZ complex and SNARE proteins [343,360] (but not under all conditions [8]) and the maintenance of normal ER morphology [8]. As described in Section 3.1.1, Rab18 has also recently been shown to promote anterograde ER tubule transport via its interaction with kinectin-1 [198] and in agreement with this result, the dynamic tubular regions of the network were absent in cells depleted of Rab18 [8]. 

A similar relationship between morphology-regulating proteins, ER dynamics, and disease is observed with ataxin-2 in spinocerebellar ataxia type 2 (SCA2). Mutations in ataxin-2 are known to cause SCA2 [350]. Depletion of ataxin-2 increased the proportion of sheets in the ER network as well as hindering both tubular and lumenal dynamics [28]. Unidirectional, long-distance translocation of tubules was diminished and FRAP experiments showed that the transport of lumenal components was much slower in the absence of ataxin-2 [28]. 

Mutations in Rab18 and ataxin-2, proteins known to play a role in extending ER tubules, both cause defects in tubular morphology as well as dynamics. Alongside membrane-shaping proteins and proteins involved in ER-late endosome/ lysosome MCSs, another likely cause for diseases is the miscommunication between the ER and mitochondria. ER–mitochondria MCS are important sites of Ca2+ ion transfer [275]. Impaired calcium homeostasis was first proposed as a possible cause of neurodegenerative diseases by Khachaturian in 1994 [361]. 

Since then, mutations in many proteins regulating ER-mitochondria MCS have been implicated in human diseases. Mutations have been found in six proteins that are either recruited to, or involved in forming ER–mitochondrial MCSs in Parkinson's disease: Miro1 [318], Parkin [321], DJ-1 [323], α-synuclein [325], PINK1 [327], and LRRK2 [329]. These mutations were all found to alter calcium ion transfer between the organelles, disrupt ER–mitochondrial MCS, or both (see Table 1). It is important to note that Miro1 also recruits kinesin-1 to mitochondria, and KIF5A mutations have been shown to affect mitochondrial motility [362]. In Alzheimer's disease, mutations in presenilins also perturb calcium ion homeostasis [335]. 

In this case, however, it is the operation of ryanodine receptors (RyR) and inositol 1,4,5-triphosphate receptors (IP3R) that are dysregulated, rather than the MCSs [333,334,363]. Charcot Marie Tooth (CMT) disease has also been linked to mutations in ER-mitochondria MCS-forming proteins [303,306], although no link has been found between CMT and calcium dynamics. 

The dysregulation of membrane contact sites between the ER and mitochondria has also been implicated in other non-neurodegenerative diseases. Impaired insulin signaling, a hallmark of diabetes, is observed in cells with mutations in mitofusin 2 [308,309] and the VDAC-1/grp75/IP3R-1 Ca2+ transfer complex [310], both of which tether the ER and mitochondria at MCSs. 

Additionally, many proteins at the ER-mitochondria interface have been implicated in various cancers (reviewed in [345]). Abnormal mitochondrial fission at ER–ER-mitochondrial MCSs due to mutations in dynamin-related protein 1 also leads to encephalopathy [340]. Additionally, ER–mitochondrial MCSs have been linked to ALS via mutations in the sigma-1 receptor [300], which is involved in Ca2+ homeostasis at MCSs. These MCSs were disrupted in mutant cells [302]. ALS mutations have also been linked to altered Ca2+ dynamics at ER–mitochondrial MCSs. The ALS mutant form of VAPB leads to an altered MCS with the outer mitochondrial membrane protein PTPIP51, perturbing Ca2+ uptake at these MCSs [296]. 

Many other ALS mutations affect ER–mitochondrial contacts, such as those in VAPB (see reviews by Chen et al. and Borgese et al. in this issue [275,291]). Intracellular calcium homeostasis also involves MCSs between the ER and the plasma membrane. Upon depletion of ER calcium stores, extracellular Ca2+ is transferred across the plasma membrane into the ER at MCS via SOCE. 

The ER-resident protein STIM1 and the plasma membrane-resident protein Orai1 form a complex, facilitating SOCE (see Section 2.2.4). Mutations in either or both of these proteins are known to cause tubular aggregate myopathy (TAM) and the related condition Stormorken syndrome via dysregulation of calcium homeostasis [311–315]. Since STIM1 binding to EB proteins generates TACs, it will be interesting to determine the effects these mutations have on TAC-based ER tubule extension. Pathogenic infections are also linked to ER-related proteins. 

Pathogens hijack the ER in the host cell to further their replication (reviewed in [364,365]). For several of these pathogens, proteins have been discovered that bind to ER-resident proteins to form MCSs. These sites are crucial for pathogen replication. 

Known ER-interacting pathogens include Legionella pneumophila [352], Brome mosaic virus (BMV) [356], the non-enveloped polyomavirus SV40 [206], enterovirus 71 [357], flaviviruses such as the Zika virus [358], and Chlamydia trachomatis. Reticulons are targeted by Legionella pneumophila via the binding of Ceg9 [352], BMV via viral protein 1a [356], and enterovirus 71 via enterovirus protein 2C [357]. 

Pathogen replication is promoted by the binding of reticulons in all three of these cases [351,356,357]. Legionella pneumophila also interacts with atlastins [352] to promote replication [351], as do the flavivirus family members: Dengue virus, Zika virus, and West Nile virus [358]. Chlamydia trachomatis proteins bind to VAPA, VAPB [353], and CERT [354] to form membrane contact sites with the ER. 

Depletion of CERT or the VAP proteins diminished bacterial replication [354], demonstrating once again that ER-pathogen MCSs are critical for pathogen replication. Interestingly, ER membrane-shaping proteins have also been found to suppress viral replication. Reticulon 3 was found to bind to nonstructural protein 4B (NS4B) of the hepatitis C virus [366]. The self-oligomerization of NS4B facilitates viral replication [367]. 

However, when reticulon 3 is bound, self-oligomerization of NS4B is prevented, and therefore viral replication is suppressed [366]. SV40 enters the cytoplasm by penetrating through the ER membrane using the ERAD machinery [368], most likely in an expanded perinuclear ERQC (Section 2.2.1), and this has been suggested to involve kinesin-1 function via binding to B14, an ER-localised DNA-J domain-containing membrane protein [206]. 

In summary, mutations in single proteins can have catastrophic effects on ER morphology, dynamics, and MCSs, leading to clinical pathologies. In many cases, morphology, dynamics, and MCSs are tightly linked, with mutations affecting one of these three attributes often causing knock-on effects for the others. Recent research into disease-causing mutations is beginning to consider ER morphology and dynamics as related topics, as opposed to viewing them independently. 

Future work in this area is likely to provide insights into the relationship between morphology and dynamics as well as how ER dynamics are involved in diseases that are known to alter morphology.

5. Discussion

Many proteins that are responsible for regulating ER morphology have been identified and the links between morphology and function are now becoming clear. We are only just beginning to quantify ER dynamics and to understand what effect the dynamics may have on the processes performed by the organelle. 

Mutations in morphology-regulating proteins have been found to cause many human diseases (Table 1) and in many cases, the structure of the ER was abnormal, indicating that the morphology, dynamics, and function of the ER are intrinsically linked. ER dynamics are also clearly vitally important in establishing and maintaining the organization of the ER and its many MCSs. Again, the proteins involved are commonly mutated in disease. A key challenge for the future is to define the roles played by these dynamics and tease apart the contribution of different kinds of motility. 

This will need faster frame rate imaging that has commonly been used, since highly motile ER tubules and potentially interacting organelles move at speeds of up to 5 µm/s, and cannot be reliably detected when imaging at one frame every 1.5–5 s, as have commonly been used (e.g., [22,38,75,176,219]). It is also important to note that the go-to cell line for studying ER dynamics, the Cos-7 cell, has a much less dynamic ER than other cell lines [20]. 

The advent of super-resolution microscopy of fixed and live cells will continue to provide a wealth of new understanding of ER structure and dynamics. We expect that with the recent publication of several methods to analyze and quantify ER dynamics, more links between dysregulated ER dynamics and disease will be discovered. Hopefully, these discoveries will result in a further understanding of the functional basis for ER movement. The future of ER dynamics research looks bright!
Author Contributions: H.T.P. and V.A. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding: H.T.P. was funded by an Engineering and Physics Research Council Ph.D. DTP studentship, grant number EP/N509565/1.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: Not applicable.

Acknowledgments: This review has covered a highly complex topic, and we would like to apologize to our colleagues whose work we were not able to include.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the writing of the manuscript, or in the decision to publish the results.

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