Biological Basis of Cell Trafficking: A General Overview

ABSTRACT

Cell trafficking is a tightly regulated biological process for the exchange of signals and metabolites between cell compartments, including four main processes: membrane trafficking (transport of membrane‐bound vesicles), autophagy, transport along the cytoskeleton, and membrane contact sites. These processes are cross‐sectional to cellular functions, ranging from the transportation of membrane proteins, membranes, and organelles to the elimination of damaged proteins and organelles. In consequence, cell trafficking is crucial for cell survival and homeostasis, serving as a cornerstone for cellular communication and facilitating interactions both with the surrounding environment and between different organelles. Disorders of cell trafficking are clinically and pathophysiological diverse and complex and form the largest group in the recent International Classification of Inherited Metabolic Disorders (ICIMD). In this review, we explore the four categories of cell trafficking and the biological principles that drive these processes. Instead of delving profoundly into each pathway, as comprehensive reviews on those topics already exist, we offer a broad overview of the molecular mechanisms behind cell trafficking, providing a foundational understanding to ease their entry into this subject and enhance comprehension of the other articles featured in this Special Issue.

1. Introduction

Cell trafficking is the biological process for the exchange of signals and metabolites between cell compartments. Under this definition four main processes are included: membrane trafficking (transport of membrane‐bound vesicles), autophagy, transport along the cytoskeleton, and membrane contact sites. As in most cellular activities, these four processes are not independent and isolated but are interrelated and coordinated with each other. This is made evident at numerous levels, from the interconnection of the different pathways themselves (e.g., vesicles are transported from one compartment to another through the cytoskeleton, which also plays a key role in vesicles scission) to the organelles involved in these processes (endoplasmic reticulum and Golgi play central roles in membrane trafficking and are also involved in autophagy initiation) or in the protein complexes involved, which many times are shared through different cell trafficking pathways.

The proper functioning of these pathways is essential for cellular homeostasis, as the study of the pathologies resulting from alterations in cell trafficking shows. The number of monogenic diseases of cell trafficking has increased over 7 folds in the last 10 years, forming the largest group in the recent International Classification of Inherited Metabolic Disorders (ICIMD) [1]. While from the perspective of inherited errors of metabolism (IEMs) only membrane trafficking and autophagy fall within this category of diseases, disorders of both transport through the cytoskeleton and the membrane contact sites (MCS) fulfill the criteria to be considered under this group and could be included in a future updated classification.

Trafficking disorders usually hold complex pathophysiological mechanisms that include alterations in various cellular processes, which need to be studied for their correct treatment. The vast majority of these diseases affect many organs, as reflected by the heterogeneity and complexity of the clinical presentations. Despite this plurality, cell trafficking disorders share an outstanding compromise of the nervous system, including both early neurodevelopmental encephalopathies and neurodegenerative disorders. On top of being the primary cause of disease, cell trafficking alterations also underlie in the pathophysiology of other frequent disorders such as Parkinson or cancer, which accentuates the interest and importance of their study.

In this review we aim to walk through the four categories of cell trafficking and the biological basis underlying these processes. The four processes and main players have been summarized in Figure 1 and Table 1, respectively. Rather than deepening into each pathway, for which other excellent specific reviews have been published, we will provide a general view of the molecular basis of cell trafficking. This will provide an initial approach to the subject and facilitate the understanding of the rest of the articles included in this Special Issue. Only some of the most representative disorders are here mentioned since a detailed table containing a wide range of disorders of cell trafficking is included in the article of this special issue: “Trafficking disorders: phenotypical similarities and differences with other IMDs” (García‐Cazorla et al).

FIGURE 1.

Membrane trafficking pathways and proteins involved. Schematic representation of the four categories of cell trafficking: Membrane trafficking related to endocytosis (pink) and exocytosis (purple), autophagy (blue), trafficking along cytoskeleton (light blue), and membrane contact sites (yellow). Main proteins involved in each pathway are colorized in green: CORVET, ESCRTs, HOPS, Retromer, and Retriever in endocytosis; COPI and COPII in exocytosis; Dynein and Kinesin in cytoskeleton transport. ERES (ER exit sites), ERGIC (ER‐Golgi intermediate compartment).

TABLE 1.

Main protein complexes of cell trafficking.

Protein complex	Definition and process involved
COATOMERS	Essential for membrane trafficking. Involved in both cargo selection and lipid bilayer deformation, leading to the new vesicle formation
COP II	Forward trafficking (RE‐Golgi). Binds to the membrane and makes the curvature for budding. Formed by core proteins (Sec12, Sar1‐GTP, Sec23/24, Sec13/3) + accessory proteins (Sec16A, Tango1)
COP I	Retrograde transport (Golgi‐RE + intra TGN‐CGN). Formed by the core proteins (Arf‐1, Arf‐GAP, Arf‐GEF, and coatomer)
ENDOSOMAL PROTEIN SORTING	Endosomal membrane protein complexes that select the cargos to be recycled. The whole process requires the main complexes + accessory proteins
RETROMER	Protein complex consisting of the vacuolar protein sorting members VPS26, VPS29, and VPS35. It is recruited to the membrane by SNX2 and Rab7. SNX27 and SNX3 bind the cargo to the retromer, and SNX‐BAR proteins deform the membrane for tubulation, together with the WASH complex
RETRIEVER	Similar in function to Retromer (and overlaps in the recycling of certain substrates). Composed of DSCR3, C16orf62, and VPS29. Associates with SNX17 and couples to CCC and WASH complexes
ENDOSOMAL SORTING COMPLEXES REQUIRED FOR TRANSPORT (ESCRT)	Group of protein complexes that associate with membranes to drive their deformation away from the cytosol, either in intraluminal vesicles or to the extracellular space
ESCRT‐0	Recognition and binding of ubiquitinated proteins to the endosome
ESCRT‐I	Clusters Ub‐proteins by bridging between ESCRT‐0 and ESCRT‐II. Some key members are VPS23, VSP28, VSP31, and MVB 12
ESCRT‐II	Key members are VPS22, VPS25, and VPS36
ESCRT‐III
ENDOCYTIC TETHERING COMPLEXES	Protein complexes that participate in tethering the membranes of the different compartments of the endolysosomal pathway. They share 4 of the 6 subunits: VPS18, VPS11, VPS16, and VPS33A
CORVET	Two subunits add to the shared core: VPS8 and VPS3. Participates in the earliest steps
HOPS	Two subunits add to the shared core: VPS41 and VPS39. Participates in the latest steps of the endolysosomal pathway

Note: Principal complexes are highlighted in yellow with their components above. The right columns describe the characteristics, subunits, and functions of each protein complex.

2. Membrane Trafficking

Membrane trafficking is the movement of membrane‐bound vesicles throughout the cell, allowing the exchange of proteins, macromolecules, and membranes themselves between organelles, as well as the entry and exit from the cell. This includes, but is not limited to, two processes: exocytosis and endocytosis.

EXOCYTOSIS refers to the movement of vesicles to the outside of the cell. It vertebrates the transport of lipids and of membrane proteins (which constitute over 30% of the total proteome) to their destination and the delivery of vesicle contents to the extracellular space. The process starts at the Endoplasmic Reticulum (ER), particularly in the specialized site within the ER named Endoplasmic Reticulum Exit Sites (ERES), where the vesicle containing the cargo is formed. This process requires the participation of the COP‐II coatomer, a complex of vesicle‐coat proteins that enable the formation of the vesicle. It assembles into a coat, selecting and binding the cargo proteins and inducing the membrane curvature that will lead to vesicle budding. This complex is formed by four subunits (Sec12, Sar1‐GTP, Sec23/24, and Sec 13/3) all of which are necessary but not sufficient for vesicle formation, as the cooperation of ERES‐resident proteins (such as Sec16A and TANGO1) is needed. Upon cargo selection and formation of the coatomer, the complex polymerizes into a curved scaffold, supporting membrane deformation and finally protein sorting in a vesicle that will be transported to the Golgi System (GS). Recent studies complement this traditional model in which ERES are a specific limited area of the ER in which a vesicle is formed and travels from the ER to the GS, proposing that in some cell types, the ERES will form an interwoven tubular continuous network for the protein export, extending these protrusions along microtubules while attached to the ER by a thin neck [2].

Once sorted from the ER, the vesicles travel through a membranous tubule‐vesicular organelle referred to as the Endoplasmic Reticulum‐to‐Golgi Intermediate Compartment (ERGIC), located close to the ER. It mediates the traffic of the cargo between the ER and the Golgi system. Two different models have been proposed to explain its dynamic nature regarding the secretory pathway: the “transport complex” and the “stable compartments” models. In the first one, ERGIC is described as a transient structure that results from the fusion of the ER‐released vesicles that migrates to form the cis‐Golgi system. On the other hand, the latest defines the ERGIC as a stationary organelle with its own identity that gathers a cargo from the ER and releases the vesicle to the cis‐Golgi. Both models can co‐exist, at least in non‐polarized cells and in dendrites, in which the predominance is of stationary structures and mobile elements move in a stop‐and‐go fashion [3].

Upon arrival to the cis‐Golgi network (CGN), the transported protein undergoes a series of post‐translational modifications before departing to its destination. Three models describe cargo trafficking through the GS: the cisternal maturation, the rapid partitioning, and the Golgi tubule‐mediated transport model [4]. The cisternal maturation model proposes that the secretory cargo moves in an anterograde direction with the whole cisternae, which mature from the cis‐ to the trans‐Golgi face, while GS‐resident proteins are transported retrogradely in COPI vesicles. Opposite to that, the rapid partitioning model proposes that GS cisternae are divided into subdomains based on their lipid composition, with secretory proteins concentrated in an export domain and GS‐resident proteins in a processing one. Finally, the Golgi tubule‐mediated transport model suggests that each cisterna is connected by tubules regulated by the COPI complex, facilitating rapid bidirectional transport within the Golgi complex. Despite the importance of GS as a hub for membrane trafficking, intra‐Golgi trafficking mediators are still under definition. From the Golgi, the cargo integrated in a membrane will depart to its final destination.

Opposite to the secretory pathway, ENDOCYTOSIS refers to the incorporation of a substance into the cell through the formation of a vesicle from the plasma membrane enveloping the cargo. It is essential for spatiotemporal regulation of signal transmission and communication with the environment and is involved in processes as relevant for neuronal function as termination of signaling through receptor downmodulation, as reviewed in [5]. Basic mechanisms of endocytosis include micropinocytosis, clathrin‐mediated, and clathrin‐independent endocytosis among others, and they define the type of vesicle formed. The very process of membrane invagination and cleavage of the vesicle from the plasma membrane is complex and requires the interaction of multiple agents, as reviewed in [6].

Upon internalization, the incorporated vesicle fuses with the Early Endosomes (EE), the first compartment of the endocytic pathway. They function as primary hubs for the sorting of the endocytosed cargo, which fate can diverge in three different routes: (a) rapid recycling, whereby the cargo is returned directly to the membrane; (b) retrograde transport, which allows internalization from the EE to the trans‐Golgi Network (TGN); or (c) maturation of the endosome by the endolysosomal pathway, in which late endosomes fuse with lysosomes, degrading the vesicle content (see Figure 1). All three pathways depart from the EE compartment, where the cargo is selectively sorted through membrane remodeling events. Rather than different populations of vesicles, early endosomes are membranous organelles in which both opposing pathways can occur parallelly by spatial separation of distinct “degradative” and “recycling” domains [7, 8].

The first two paths, rapid recycling and transport to the TGN, allow cargo recovery, avoiding its degradation in the lysosome. Some of the proteins sorted in this pathway are transmembrane proteins and receptors, processing enzymes, SNARE proteins, and even some toxins. Retrograde transport of lipids and proteins from the endosomes to the TGN is essential for membrane homeostasis and to counterbalance anterograde transport by recovering its components. Two highly conserved multiprotein complexes orchestrate recycling and retrograde transport from the EE: Retromer and Retriever [9]. They are endosomal coat complexes that specifically recognize and sort the cargo within the EE subdomains, clustering and forming tubulovesicular carriers for its transport to their target membranes. Both complexes differ in composition but are structural and functionally analogous. As reviewed by [10], some cargoes are specific for Retromer (such as the glucose transporter GLUT1), some for Retriever (Integrin b1), and some require co‐operation between the two sorting complexes (e.g., CD97). Other mechanisms can also operate on cargo sorting, such as clathrin adaptors like AP1 or EpsinR, Sorting Nexin (SNX) complexes, or AP‐5 (which does not operate at the EE but at the late endosome). From the GS, retrograde transport can follow to the ER. This Golgi‐Reticulum retrograde transport occurs via COPI‐coated vesicles, which bud from the Golgi and carry cargo proteins and lipids back to the ER, ensuring the continuous flow of materials between these organelles. As the aforementioned COPII, COPI is a protein complex formed by several subunits that form an inner and an outer coat that participate in cargo sorting and vesicle formation [4].

Unless diverted through rapid recycling or retrograde transport, the early endosomes will undergo the endolysosomal degradative pathway, ending up in the cargo degradation in the lysosomal compartment. The EE matures into late endosomes (LE) and eventually into multivesicular bodies (MVB). This process requires the recruitment of key proteins from the Endosomal Sorting Complexes Required for Transport (ESCRTs), which sort ubiquitinated proteins and lipids in intraluminal vesicles (ILVs), producing the MVB. As reviewed in [11], ESCRTs are three different multimeric protein complexes (ESCRT‐ I, II, and III) that are consecutively recruited from the cytoplasm to the endosomal membrane [12]. Briefly, ESCRT‐I recognizes the ubiquitinated cargo and binds to ESCRT‐II, which activates and recruits ECRT‐III, driving membrane remodeling and scission. The MVB will be further fused with a lysosome, degrading the ILV components. Two multimeric complexes outstand in the regulation of these steps: class C core vacuole/endosome thethering (CORVET) and homotypic fusion and protein sorting (HOPS). The former is involved in early endosome fusion and maturation, regulating the tethering and subsequent fusion of endosomal compartments. Subsequently, HOPS complex regulates mainly the endolysosomal fusion steps, mediating the membranes tethering to facilitate their further fusion [13]. Mutations in the genes coding for the subunits of both complexes result in a group of neurological diseases, ranging from neurodevelopmental disorders to adult‐onset neurodegenerative diseases [14].

Membrane trafficking is a fundamental process crucial for the survival and proper functioning of all cells, but it is particularly important and complex in neuronal cells. The ultimate exponent is probably the synaptic vesicle cycle, which with its many peculiarities is essentially an ultra‐regulated cycle of exo‐ and endocytosis [15, 16]. But the importance of trafficking for neuronal function goes beyond synaptic communication. For instance, the intricate development of dendrites needs a constant influx of proteins and lipids, provided through the secretory pathway. The specialized secretory organelles (ER and Golgi) show different features to adapt to the high demands of neuronal trafficking. While the ER and functional ERES span throughout the entire neuron, the Golgi apparatus is enriched within the somatodendritic compartment. Yet distally dendritic branches count on a widespread Golgi‐related satellite microcompartment, suggesting that locally synthesized transmembrane proteins may use a direct ER‐ERGIC‐Golgi‐plasma membrane pathway [17]. As recently reviewed [18], mutations in the genes affecting the protein machinery involved in cell trafficking yield to different diseases with multiple presentations, within which outstand a compromise in neurodevelopment, either in an early or late onset. Alterations can occur in any of the processes described, from forward ER‐GS transport (as mutations affecting proteins as COP, SEC, or TRAPPC) [19, 20] to mutations affecting the GS‐ER retrograde transport (as mutations in the NRZ complex [21]).

3. Autophagy

Autophagy is the fundamental process through which the cell recycles its components, thus playing a pivotal role in cellular homeostasis. In summary, the degradative process starts with the signaling of a molecule to be degraded in the cytoplasm, followed by the nucleation of a phagophore around the cargo. Subsequently, the membrane of the phagophore elongates, facilitating its expansion and closure forming a double‐membraned vesicle named autophagosome. Upon completion, autophagosomes undergo maturation through fusion with lysosomes, forming autolysosomes, where the cargo is degraded by lysosomal hydrolases. In the interconnected network of cell trafficking, this orchestrated sequence of events ensures the efficient removal of cytoplasmic components and damaged proteins, serving as a crucial cellular mechanism for maintaining homeostasis and responding to stress conditions. Each step involves the coordinated activity of many players, which are traditionally used as markers for the study of autophagy. Since autophagy and its importance in cell homeostasis has excellently been reviewed from all possible angles, including molecular basis [22, 23] and disease perspective [24, 25], in this work we will just provide a general overview to frame its importance within Inherited Metabolic Disorders and cell trafficking.

The importance of autophagy in cellular homeostasis becomes clear when we study its role in the pathophysiology of diseases. Primary dysfunction of autophagy due to mutations in any of the genes coding for the autophagy‐core players or closely related proteins lead to a great group of diseases recently referred to as “autophagopathies” [26]. They can appear due to defects in any step of the autophagy process and be related to any of the functions of autophagy beyond the classical degradative macro‐autophagy, such as LC3‐associated phagocytosis, exosome and protein secretion or retromer‐dependent trafficking, as reviewed by [25]. On top of that, dysregulation of autophagy has been implicated in the pathogenesis of numerous metabolic and non‐primary metabolic diseases, such as neurodegenerative disorders [27, 28] or cancer [29, 30].

Autophagy defects form a sub‐group of Inherited Metabolic Diseases in the recent IMD Classification [1]. They can present as early onset (usually with neurological, neuromuscular, and multisystem manifestations) and/or late onset, in which neurodegeneration overcomes the neurodevelopmental disorder. An illustrative example of these disorders is WDR45‐related neurodevelopmental disorder, also known as BPAN (MIM # 300894). It results from mutations affecting WDR45, an autophagy‐related protein involved in the formation of the autophagosome. Its clinical spectrum is variable and includes the mentioned biphasic evolution, characterized by early onset epileptic encephalopathy followed by a progressive dystonia‐parkinsonism phenotype, together with intellectual delay and cognitive decline during early adulthood [31]. This biphasic character highlights the importance of autophagy in both neurodevelopment and neuronal maintenance [24].

In neuronal cells, much like membrane trafficking, autophagy holds particular significance. Removal and turnover of damaged organelle and proteins are critical in neurons, given their postmitotic nature and longevity, together with the high metabolic demand to support their complex function and synaptic activity. Moreover, it also plays a pivotal role in the regulation of synaptic plasticity, by removing dysfunctional synaptic components, and synaptic homeostasis, by facilitating the turnover of synaptic vesicles [32]. For instance, local autophagosome biogenesis (that occurs near the synapse) is coupled to neuronal activity [33, 34, 35, 36]. Several players involved in vesicle endocytosis also have roles as autophagy regulators, such as synaptojanin, EndophilinA [37], or the recently described Synaptogirin [38]. ATG9 has been lately proposed as one of the main agents coupling presynaptic autophagy to the synaptic vesicle cycle [39], even though it does not colocalize with synaptic vesicles themselves [40]. According to this model, ATG9 vesicles are formed from the trans‐Golgi network (located at the cell body) and delivered to presynaptic sites, where they undergo exo‐endocytosis cycle in a synaptic‐activity dependent manner, coupling autophagosome biogenesis at presynaptic sites with the activity‐dependent synaptic vesicle cycle. The synaptic terminal lacks the machinery for autophagosome degradation. As reviewed by [32], the autophagosome moves through retrograde transport toward the soma, maturing to fully degradative autolysosomes as they move along the axon [35]. This process is one of the many examples of the involvement of the cytoskeleton in cell trafficking, especially relevant in polarized cells as neurons, where vesicles have to travel through long distances.

4. Transport Through the Cytoskeleton

The cytoskeleton is a dynamic network of protein filaments extending through the cytoplasm and scaffolding cellular structure. As reviewed by Kast and Dominguez [41], it plays key roles in most forms of intracellular trafficking, from promoting the biogenesis of vesicles to the transport of cargoes throughout the cell. To such end, several proteins intervene, including the protein families forming the cytoskeleton (tubulin, forming microtubules, and actin filaments), but also motor proteins, such as dynein and kinesin, responsible for anterograde and retrograde transport, respectively, and adaptor proteins, that bridge between cargo molecules and motor proteins. Besides transport itself, cytoskeleton is crucial in cell trafficking, as it is involved in the biogenesis of vesicles as well. As reviewed by [41], actin polymerization is necessary for the biogenesis of autophagosomes from the ER membrane. Lately, mature autophagosomes detach from the ER membrane, undergo intracellular transport, and then fuse with lysosomes, endosomes, and multivesicular bodies through mechanisms that involve the cytoskeleton.

Transport through the cytoskeleton is particularly relevant in the context of neuronal function, as the cargoes (such as vesicles or mitochondria) need to travel through the axonic large distances. Turning again to synaptic metabolism, transport is directly related to the synaptic vesicle cycle. All the protein and membrane synthesis machinery is located in the neuronal soma, where scaffolding proteins, synaptic vesicle proteins, ion‐gated channels, etc., are synthesized. Transport from the soma to the synaptic vesicle (i.e., anterograde transport) is essential for the delivery of such synaptic precursors. Upon synthesis, they are next transported in defined stoichiometric ratios to the synaptic terminal, along with membrane precursor, which evidence suggests that it originated in the TGN with the participation of Rab2 [42]. Several proteins intervene in this anterograde transport, such as kinesins (that move along the microtubule) and adaptor proteins such as Arl8, an Arf‐like GTPase that also intervenes in the transport of lysosomes and synaptic vesicle precursors. The importance is that in highly active synaptic terminals, presynaptic microtubules can be rate‐limiting for neurotransmission due to the replenishment of synaptic vesicles among others [43, 44].

Retrograde transport, that is, from the synaptic vesicle to the soma, is also essential for the maintenance of synaptic homeostasis. Mutations impacting vesicle transport along microtubules, like those affecting kinesins and dynein, result in various developmental neuropathies [45, 46]. As a representative example, mutations affecting the function of kinesins have been related to Hereditary spastic paraplegia (HSP), a group of diseases characterized by progressive spasticity, Charcot–Marie–Tooth (CMT), and ALS. Around 10% of them are due to mutations in KIF5A (MIM # 617235, 604 187, 617 921), responsible for the transport of different cargoes including lysosomes.

5. Membrane Contact Sites

Membrane contact sites (MCS) are specialized subcellular structures where the membranes of two organelles come into close proximity, enabling direct communication and the exchange of ions, lipids, and other signaling molecules. As recently reviewed [47], MCS have changed the paradigm on how organelles operate together, moving from separated organelles that only communicate by vesicle transport to functional close contacts between them. The organelles do not strictly fuse but are close enough to be tethered at 30–50 nm from each other, accomplished by tethering proteins, that bring both membranes together and bridge between them. Depending on the nature of the organelles, the contacts can be homotypic (between identical organelles) or heterotypic (between two different organelles or two different membrane types) [48]. Moreover, some interactions can implicate three organelles or two regions of the same organelle, called intraorganelle contact sites. Extensive reviews on MCS can be found in [47, 48, 49].

One of the most studied examples of MCS is the contact between ER and mitochondria. Both organelles form extensive interactions termed mitochondria‐associated membranes (MAMs). MAMs are involved in lipid transfer, calcium signaling, the regulation of mitochondrial dynamics, and bioenergetics. Dysfunction of MAMs has been implicated in numerous diseases, including neurodegenerative disorders, metabolic syndromes, and cancer. For example, mutations in Dynamin‐related Protein 1 (Drp1) promote mitochondria fragmentation in Huntington's disease (HD), a neurodegenerative disorder with a selective loss of striatal neurons. Consequently, mitochondria and ER contact is disrupted, decreasing Ca²⁺ influx and increasing mitochondria superoxide species production in mice models [50].

Besides contacts with the ER, mitochondria also interact with a wide range of organelles. For instance, peroxisomes and mitochondria both use the same factors and enzymes for their division, reinforcing the crosstalk between both organelles. The interaction between both organelles, along with the RE, has been proposed as the principal mechanism for lipids delivery to the mitochondria. To such end, direct interaction of mitochondria and lipid droplets‐membrane proteins has also been described, suggesting the formation of a contact site that facilitates the transport of lipids. Besides the ionic and metabolite transfer, mitochondrial contact sites can also be crucial for quality control. Such is the case of mitochondria‐lysosome interaction, which not only mediates lipids, calcium, and ions exchange but also regulates mitophagy and autophagy. Proteins involved in these processes, such as Rab7 and GDAP1, have been suggested as regulators of mitochondria‐lysosome contact sites, based on the pathophysiological mechanisms of mutations in both genes, which result in different types of Charcot–Marie–Tooth disease (Rab 7 MIM # 600882 and GDAP1 MIM # 214400, 608 340, 607 706, 607 831). Due to Rab7 GTP hydrolysis impairment, mitochondria‐lysosome contact sites are prolonged in Rab7 mutants, causing mitochondria fragmentation and affecting its dynamics [51, 52]. In the last few years, Membrane Contact Sites between mitochondria and nuclear membrane have been gathering attention, as recently reviewed in [53]. They have a key role in the regulation of gene expression and cellular response to stress and damage, as well as intervening in the regulation of nuclear metabolism.

Metabolic pathways at MCS are tightly regulated and play critical roles such as Ca²⁺ homeostasis, lipids metabolism and transfer, and regulation of organelle dynamics. All these processes are crucial for cell survival, and in consequence, alterations in MCS lead to several disorders. As expected, the pathophysiology of these diseases includes multiple organelle dysfunction. For example, mitochondria‐lysosome contact site mediates calcium influx into the former through the ion channel mucolipin (TRPML1, encoded by the gene MCOLN1). Mutations in MCOLN1 result in the lysosomal storage disorder mucolipidosis type IV (MIM # 252650) and encompass defects on both mitochondrial and lysosome functions. Not only mitochondrial‐lysosome contact sites lead to pathological situations; the recently described connection between mitochondria and nucleus is disrupted in LONP1 deficiency causing Cerebral Ocular Dental Auricular Skeletal Anomalies Syndrome (CODAS) (MIM # 600373). This protein is involved in mtDNA maintenance and in some situations localizes to the nucleus, suggesting the connection between both cell structures [54].

6. Altered Autophagy as a Common Trait in Cell Trafficking Disorders

As aforementioned, a growing number of studies describe alterations in autophagy underlying the pathophysiology of different cell trafficking diseases, which we will briefly review through specific examples through this section. Due to our knowledge on autophagy modulation, studying the place it takes within cell trafficking disorders pathophysiology can potentially lead to the description of new therapeutic strategies.

6.1. Autophagy in Disorders of Membrane Trafficking

Autophagy is closely related with the processes of exo‐ and endocytosis, not only functionally but also at a structural level, with the orchestrated participation of several shared proteins and organelles. Therefore, it comes with no surprise the description of autophagy as part of the pathophysiology of membrane‐trafficking defects.

EXOCYTOSIS: The process of transport of a newly synthesized protein starts with the trafficking from the endoplasmic reticulum (ER) to the Golgi System (GS). One of the coordinators of this transport is TANGO2, involved in ER‐GS transport and lipids metabolism [55, 56]. Pathogenic variants in TANGO2 (MIM #616878) lead to pathology with poor prognosis, characterized by intellectual disability, seizures, and fasting‐precipitated rhabdomyolysis due to acute breakdown of skeletal myofibers [57]. Besides the alterations in endomembrane trafficking [56], TANGO2‐patient myoblasts show reduced LC3II levels in starving conditions, suggesting an impairment in autophagy along with the trafficking defects [58].

Another example of the involvement of autophagy in the pathophysiology of exocytosis defects is TRAPPC11 deficiency (MIM #615356). TRAPPC11 is a protein involved in the earliest steps of ER‐GS transport [59], and mutations affecting its function are founders of a congenital defect of glycosylation [60] characterized by muscular dystrophy [61]. Even though its main function is related to membrane trafficking, it has been described that TRAPPC11 has a role upstream of autophagosome formation. Upon TRAPPC11 depletion, LC3‐positive membranes accumulate before and during starvation but fail to be cleared. Moreover, it has been described that TRAPPC11 recruits ATG2 and WIPI4, both of which are proteins directly related to the isolation of membranes necessary for autophagy [62]. The involvement of autophagy in the pathophysiology of this disease outlines a potential therapeutic strategy.

ENDOCYTOSIS: The overlap between endocytosis and autophagy is included in the very own process of vesicle internalization since one of the new vesicle possible destinations is the endolysosomal pathway that converges with autophagy. Impairments have been described as underlying in the pathophysiology affecting the endolysosomal pathway. One example is the defects affecting the mentioned HOPS complex, which result in a group of neurodevelopmental diseases referred to as HOPSANDS [14], characterized by generalized inherited dystonia with brain iron accumulation. Interestingly, functional studies in HOPS patients' fibroblasts (due to mutations in VPS16, VPS41, or VPS11 genes; MIM # 619291, 619 389, 619 637, and 616 683, respectively) showed clear abnormalities in the lysosomal and autophagic compartments characterized by increased expression of autophagosome markers (p62/SQSTM1, LC3B) and lysosomal and hydrolases markers (such as LAMP1, b‐glucocerebrosidase, acid a‐glucosidase, and acid ceramidase). Both results suggest an accumulation of autophagosomes and lysosomal components, which are linked with large vacuolar structures detected by electron microscopy [63].

6.2. Autophagy in Defects of Transport Along the Cytoskeleton

As previously explained, cytoskeleton plays key roles in most forms of intracellular trafficking, from promoting the biogenesis of vesicles to the transport of cargoes throughout the cell. It is essential in autophagy progression, as maturing autophagosomes are transported along microtubules to perinuclear region, where the cellular concentration of lysosomes and late endosomes is highest [35]. This takes special relevance in highly polarized cells as neurons, where autophagy for synaptic health maintenance takes a two‐way trip. While autophagy is notably active in the presynaptic bouton, autophagosome biogenesis occurs in the distal axon, and lysosomal degradation is confined mainly to the soma. Therefore, autophagosomes need to be retrogradely transported to the cell body, maturing along their way [64]. This transport is mediated by dyneins [65, 66] and is evidenced by the gradient of markers evidenced along the axon and toward the soma [35].

Functional deficits in the transport machinery are associated with a range of neurological disorders in which a role has been described. Regarding forward transport, gain‐of‐function mutations in KIF5A have been reported to cause an increase in the autophagy initiation marker p62, suggesting that an unbalanced autophagy flux contributes to its pathophysiology [67]. Not only alterations in forward transport have been related to autophagy, but retrograde transport as well. Mutations affecting the function of the dynein‐binding protein Dynactin 1 (DCTN1) cause a hereditary motor neuropathy phenotype and ALS (MIM # 105400, 607 641). Through its interaction with dynein, DCTN1 plays a critical role in retrograde transport of autophagosomes and lysosomes [68]. Animal models of DCTN1 mutants showed impaired autophagosome transport and accumulation of autophagosomes in damaged axons, suggesting an impairment in autophagy in the pathophysiology of the disease [69].

6.3. Autophagy in Defects of Membrane Contact Sites

Besides their critical roles in calcium homeostasis or lipids transfer, MCSs are also involved in autophagy regulation [49]. One of the most evident examples is the mitochondria‐lysosome MCS through GDAP1‐LAMP1. GDAP1 interacts with the lysosomal protein LAMP1, and its depletion causes membrane defects in early autophagic vesicles, lysosomes, and in their contacts with mitochondria [70], deriving in Charcot–Marie–Tooth disease.

7. Concluding Remarks

To conclude, this review offers an overview of cell trafficking processes, emphasizing the intricate coordination between them and their significance in both health and disease. The high number of cell trafficking pathologies, and its importance in the context of neurodevelopmental and neurodegenerative disorders, underscores the critical need to fully understand these processes. Delving into the complexities of cell trafficking and the pathophysiological mechanisms associated will be the base to develop innovative and targeted therapies.

Author Contributions

Helena Gimeno‐Agud: literature review and manuscript writing. Yaiza Díaz‐Osorio: literature review and manuscript writing. Alfonso Oyarzábal: review conceptualization, literature review and manuscript writing.

Conflicts of Interest

A.O. is a co‐founder of the Hospital Sant Joan de Déu start‐up “Neuroprotect Life Sciences.” The content of the article has not been influenced by the sponsors.

Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study. This review is a non‐systematic one, where literature was curated to guide toward a comprehensive overview of trafficking disorders. Key terms related to the four main trafficking processes—membrane trafficking, autophagy, cytoskeletal transport, and membrane contact sites—were used to search for relevant studies. Pathology‐related keywords were also employed to ensure the inclusion of representative data that reflect the broader landscape of trafficking disorders. The goal is to provide an understanding of the topic rather than an exhaustive examination of each specific pathway. There has not been data generation in this review.