Intertwined And Finely Balanced: Endoplasmic Reticulum Morphology, Dynamics, Function, And Diseases Part 5

3.1.2. MCS-Mediated ER Dynamics

In addition to tubule extension driven by motors directly at the ER membrane, ER dynamics can be caused by ER membrane contact sites with early and late endosomes, lysosomes, and mitochondria [22,26,38,74,75,84–86,204,219,220] that move along microtubules. 

Research has found that acid hydrolase in lysosomes can break down proteins and convert them into amino acids, which can be used by cells to synthesize new proteins. This process is important for learning and memory, as protein synthesis is closely related to the formation of memory.

In addition, lysosomes can also keep cells healthy by breaking down engulfed cellular waste and harmful substances and removing waste and toxins from the cells. This process is also critical for protecting the survival and function of neurons. Neurons are our brain cells and they play a vital role in the process of learning and memory. If neurons are affected by cellular waste and harmful substances, their function and ability to survive will be severely compromised, affecting the formation and retention of memory.

Therefore, maintaining the function and health of lysosomes is of great significance for improving memory and protecting the survival and function of neurons. How to maintain lysosome function and health? First of all, we need to pay attention to the intake of nutrients, especially the intake of important nutrients such as protein; secondly, we need to maintain adequate sleep and exercise; finally, we need to avoid bad living habits such as staying up late to reduce life stress and physical burden.

In summary, the relationship between lysosomes and memory is very close. By focusing on maintaining lysosomal function and health, we can improve memory and protect neuronal survival and function, thereby promoting the health and development of the body. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory, because Cistanche deserticola has antioxidant, anti-inflammatory, and anti-aging effects, which can help reduce oxidation and inflammatory reactions in the brain, thereby protecting the health of the nervous system. In addition, Cistanche deserticola can also promote the growth and repair of nerve cells, thus enhancing the connectivity and function of neural networks. These effects can help improve memory, learning ability, and thinking speed, and may also prevent the development of cognitive dysfunction and neurodegenerative diseases.

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This is an example of a process referred to as 'hitchhiking', where one organelle provides the motor and drives the movement of another cargo that is not motile by itself [38,221,222]. Early work on ER tubule movement identified morphologically distinct motile domains at the tips of some moving ER tubules [182,191]. 

However, there is clear evidence from transmitted light microscopy and DiOC6 labeling that ER tubules themselves can translocate directly along microtubules, without the need for hitchhiking with another organelle [19,182,183,190]. 

Around half of Rab5-positive endosomes in Cos-7 cells are attached to the ER during imaging [75], and when imaged at low frame rates (1 frame per 1.5 s) appeared less motile than ER-associated lysosomes [219], and less likely to cause ER tubule mobility [26]. 

However, live imaging at the rapid frame rates needed to capture fast early endosome movement has revealed that moving endosomes can translocate towards an ER tubule, grab it, and continue moving, pulling out an ER tubule behind them ([74]; Figure 4). 

As early endosomes move primarily towards the cell center, driven by dynein [74,223], hitchhiking on early endosomes may account for some of the dynein-dependent ER tubule extensions [20]. 

How dynein and kinesins are recruited to early endosomes, and the role of kinesin-1 in their movement, is not fully understood. Recent work suggests that 30–50% of ER tubule extension events are driven by hitchhiking on late endosomes/lysosomes moving along microtubules, while 40% of tubules moved along microtubules independently, and the remainder were mediated by TACs or dTACs (see below) [26,38,219]. 

Surprisingly, almost all lysosomes were associated with and moved with, the ER network [219]. Lysosomes that were imaged before, during, and after a hitchhiking event were seen to slow down when attached to ER tubules [204,219], possibly because there was less drag counteracting the force generated by the lysosomal motors when not extending an ER tubule. 

Two ER-resident proteins involved in phospholipid synthesis, PIS, and CEPT1, which have previously been shown to be at the tips of motile tubules along with Rab10 [7], were associated with LE/lysosome hitchhiking events [26]. 

Again, dynein and kinesin-1 are the major motors driving late endosome/lysosome movement, and there are multiple ways in which they are recruited and controlled, some of which involve interactions with the ER (reviewed in [128,129]). Dynein can be recruited via RILP and Rab7 (in the presence of high cholesterol levels); ALG2 and TRPML1 (regulated by PI(3,5)P2 and calcium); or JIP4 and TMEM55B (promoted by starvation, which increases TMEM55B transcription via mTORC1) [128,129]. Kinesin-1 is recruited by SKIP and Arl8 (regulated by BORC), or FYCO1 and Rab7 (regulated by PI(3)P and involving protrudin at the ER: see below), and both linkages are via KLCs [128,129]. 

An open question is whether these multiple mechanisms function in parallel on the same organelle, or whether there is spatial selection (e.g., on specific membrane subdomains), or switching depending on metabolic status, or other inputs. 

If only 30–50% of ER tubules move in association with late endosomes/lysosomes, how important is this ER hitchhiking for ER organization overall? Two recent studies have demonstrated that in Cos-7 cells at least, the answer is-very. 

Disrupting ER-late endosome MCSs by RNAi-mediated depletion of VAPA (which binds ORP1L to anchor LE to ER via Rab7: [128]) also disrupted ER morphology and reduced the extent of ER tubules and network complexity, especially at the periphery [26,219], with the knockdown of all ER VAP family members (VAPA, VAPB, and MOSPD2) giving a stronger phenotype [26].

In addition, manipulating the motors present on late endosomes/lysosomes provided a useful means of triggering inward or outward endosome movement, which led to a reduction or increase in peripheral ER tubules [26,219] and network dynamics [26]. 

Interestingly, the depletion of SKIP or Arl8 caused major changes in the ER network, suggesting that this is the major route for kinesin-1 recruitment for this process, rather than protrudin/FYCO1 [219]. Protrudin, the product of the ZFYVE27 gene, is a multispanning transmembrane ER protein that has a plethora of interactors, both at the ER and at late endosomes. 

At the ER, it binds to the ER shaping proteins atlastin, REEPs 1 and 5, reticulons 1, 3, and 4, and also interacts with VAP via an FFAT motif ([224]; reviewed in [225]). It can also bind to late endosomes via its Rab-binding domain, which binds Rab7-GTP, and an FYVE (Fab-1, YGL023, Vps27, and EEA1) domain, which binds to the late endosomally enriched phosphatidylinositol 3-phosphate (PI3P) [204,225]. 

Crucially, it also binds to all KIF5 family members, although the interaction is strongest with KIF5A [226], which is striking considering both protrudin and KIF5A (but not KIF5B or C) can cause hereditary spastic paraplegia when mutated (Table 1). 

Over-expression of either protein caused the formation of protrusions in non-polarised cells [226], hence protrudin's name [225], while siRNA-mediated depletion led to an expansion of CLIMP63-labelled sheet-like regions into the cell periphery [224]. Given protrudin's potential to bind to the late endosome, Raiborg and coworkers investigated if it was involved in MCS formation and found that was indeed the case [204]. 

Furthermore, overexpression of protrudin led to an accumulation of late endosomes/lysosomes at the cell periphery, a phenotype that had previously been seen for FYCO1, another PI3P and Rab7-binding protein, and which was found to interact with protrudin [204]. 

Imagining of FYCO1 and protrudin in living cells revealed that moving FYCO1-positive late endosomes interacted with protrudin at the ER, pausing or slowing down while they did this, then detached and moved off more rapidly. Protrudin binds KIF5 and FYCO1 binds KLC, and the expression of protrudin increased the amount of KIF5 found associated with FYCO1, leading to the model that kinesin is passed from protrudin to FYCO1 on the late endosome during ER-late endosome association, so activating late endosome movement once they break free of the ER. 

While this model is appealing, more formal proof is needed. What is clear though, is that both protrudin and FYCO1 are important for axon extension [129,195,204,227]. Protrudin and FYCO1-mediated late endosome translocation to the cell periphery has also been shown to be important for invadopodia formation, where late endosomes deliver the matrix protease MT1-MMP for secretion, which is necessary for cancer cell migration [205]. 

Importantly, the late endosome/lysosome position is controlled by nutritional statuses, such as cholesterol and amino acid levels, which in turn regulate dynein and kinesin-1 recruitment or activity [128,129,228]. 

This regulation has recently been shown to have a major impact on ER dynamics and distribution within the cell [26,219]. Serum starvation led to a less mobile ER network and reduced late endosome/lysosome motility, leading to a less complex ER network in the cell periphery with fewer tubule junctions [26]. Lu and coworkers found that a 4 h serum starvation led to late endosome/lysosome clustering, and a reduction in the proportion of tubular ER, as did cholesterol enrichment [219]. 

In contrast, 24-hour starvation or cholesterol depletion triggered peripheral localization of endosomes with no effect on ER tubules [219]. The protrudin-mediated ER–endosome/lysosome contact pathway is also influenced by nutritional status. The neuronal isoform of carnitine palmitoyltransferase 1, CPT1C, is an ER protein that is regulated by malonyl-CoA levels and mutated in HSP [228]. 

Recent work has revealed that CPT1C is needed for proper neuronal growth and controls the transport of late endosomes/lysosomes to the axon tip, and this needs its ability to bind to malonyl-CoA [228]. 

It interacts with protrudin, and expressing it in HeLa cells increased the proportion of outward-moving FYCO1-labelled late endosomes if malonyl-CoA was present, but reduced movement to below control levels if malonyl-CoA was depleted. 

However, unlike protrudin, CPT1C was present, but not enriched, at ER–lysosome contacts, suggesting that it regulates the protrudin–FYCO1–kinesin-1 interaction rather than being directly involved. The authors suggest that in the presence of malonyl-CoA, CPT1C promotes the transfer of kinesin-1 from protrudin to FYCO1 on late endosomes/lysosomes, thus promoting their outward movement in neurons [228]. 

However, as mentioned above, this kinesin transfer model requires further testing. Mitochondria are known to interact extensively with the ER in live cells [22], and motile mitochondria can extend ER tubules [22,38]. ER-associated mitochondria are preferentially localized to acetylated microtubules [22], which are the preferred track for kinesin-1 (e.g., [229]), which is a motor for both mitochondria (e.g., [230]) and the ER. 

Mitochondria were also seen to interact with lysosomes, and the moving lysosome could pull out a thin tubule from the mitochondrion [38]. As similar thin tubules were found to extend from mitochondria at points of ER contact via the action of KIF5B and its mitochondrial receptor Miro1 [230], it prompts the question as to whether the PDZD8-induced three-way MCSs between ER, late endosomes, and mitochondria could be involved in both processes. 

However, this complex interaction essentially immobilized the organelles [126].

MCSs are vitally important for many aspects of ER function, metabolism, and overall dynamics. This makes it challenging to interpret alterations seen following experiments designed to disrupt one aspect of MCS function. 

This is exemplified by experiments where Rab7a function-needed for late endosome/lysosome MCS, and involved in recruiting both kinesin-1 and dynein to endosomes-was disrupted. Rab7a depletion, or expression of a GDP-locked Rab7a, led to an accumulation of CLIMP63-labelled sheetlike ER at the cell periphery, and activation of the ER stress response [231]. 

Mateus et al. hypothesize that structural changes are caused by ER stress, rather than changes in ER dynamics [231]. Indeed, there are many ways in which stress can influence MCS and the ER (reviewed in [85]). Monitoring ER stress levels will be an important control in future studies (e.g., [219]).

3.1.3. Motor-Independent ER-Microtubule Interactions

It was clear from early studies that three kinds of ER–microtubule interactions exist: motor-driven translocation of ER tubules, static interactions, and the attachment of ER tubules to growing microtubule tips [23]. 

The latter interaction drives tubule extension via the formation of 'tip attachment complexes', or TACs, first seen in Xenopus egg extracts [232]. In a variety of cultured cells, motor-driven sliding was seen to predominate over TACs and static interactions between ER tubule tips and microtubules [20,21,23,38], although the percentage differed between cell types [21]. 

TACs consist of the transmembrane ER protein STIM1 (stromal interaction molecule 1), which interacts with the MT plus-end-tracking protein EB1 [21]. STIM1 interacts with Orai1 at the plasma membrane upon depletion of ER calcium stores to permit calcium transfer from outside the cell into the ER (see Section 2.2.5). Interestingly, triggering this process prevented STIM1 tip tracking [21]. 

While the presence of a TAC did not change the rate of microtubule growth and shrinkage, it reduced the likelihood of the microtubule undergoing a catastrophe (starting to depolymerize) [232]. ER tubules can also be extended by attaching to a depolymerising microtubule via a dTAC [38]; the composition of ER dTACs is not known. Why have two methods for transporting the ER towards the cell periphery? 

In the case of Xenopus embryos, although kinesin-1 is present in the ER, it is not active until later in development [187]. Instead, the ER network distributes throughout the cytoplasm via the combined activity of dynein pulling it towards the nucleus and centrosome, and TACs extending it outward as microtubules ppolymerize[184]. In cultured cells, TACs would ensure the ER reaches right to the cell edge. 

In contrast, kinesin-1's preference for stable microtubules as tracks, perhaps because they have less MAP7 bound to their surface, means it translocates poorly to the end of newly polymerized microtubules [229]. 

An important role for STIM1-EB1-mediated ER localization has been demonstrated in neuronal growth cones, where STIM1 is needed for the orientation of microtubules and ER which is essential for a growth cone to move towards a growth factor gradient [233].

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