• A variety of microfluidic device designs are widely used to study the behaviors of single bacterial cells between growth conditions, but a quantitative comparison of growth between different devices remained absent.
• The authors developed a protocol to obtain side-by-side experiments using two different microfluidic approaches, finding that growth rates and interdivision times differ but are precisely compensated by the division ratio distribution.
• Identical emergent simplicities govern stochastic intergenerational homeostasis of cell sizes across device and experimental configurations. This intercondition scaling law establishes a principled route to draw comparisons of stochastic growth and division dynamics across experimental modalities.

Microfluidic platforms enable long-term quantification of stochastic behaviors of individual bacterial cells under precisely controlled growth conditions. Yet, quantitative comparisons of physiological parameters and cell behaviors of different microorganisms in different experimental and device modalities is not available due to experiment-specific details affecting cell physiology. To rigorously assess the effects of mechanical confinement, we designed, engineered, and performed side-by-side experiments under otherwise identical conditions in the Mother Machine (with confinement) and the SChemostat (without confinement), using the latter as the ideal comparator. We established a protocol to cultivate a suitably engineered rod-shaped mutant of Caulobacter crescentus in the Mother Machine and benchmarked the differences in stochastic growth and division dynamics with respect to the SChemostat. While the single-cell growth rate distributions are remarkably similar, the mechanically confined cells in the Mother Machine experience a substantial increase in interdivision times. However, we find that the division ratio distribution precisely compensates for this increase, which in turn reflects identical emergent simplicities governing stochastic intergenerational homeostasis of cell sizes across device and experimental configurations, provided the cell sizes are appropriately mean-rescaled in each condition. Our results provide insights into the nature of the robustness of the bacterial growth and division machinery.

• We focus on patterns of a specific bacterial symbiont’s peptidoglycan (PG)-synthesis activity, a critical process supporting bacterial growth and survival from the perspectives of both developmental time (i.e., over different stages of tissue colonization) and spatial locations (i.e., between different tissue-colonization sites).
• The discovery that PG-synthesis activity varies across different stages and locations during a beneficial infection provides insights into how both mutualistic and pathogenic bacteria must adapt to, and survive long-term within, the diverse host tissue conditions.
• This knowledge will improve our grasp of bacterial persistence strategies and their roles in animal-microbe interactions.

The symbiotic relationship between the bioluminescent bacterium Vibrio fischeri and the bobtail squid Euprymna scolopes serves as a valuable system to investigate bacterial growth and peptidoglycan (PG) synthesis within animal tissues. To better understand the growth dynamics of V. fischeri in the crypts of the light-emitting organ of its juvenile host, we showed that, after the daily dawn-triggered expulsion of most of the population, the remaining symbionts rapidly proliferate for ∼6 h. At that point the population enters a period of extremely slow growth that continues throughout the night until the next dawn. Further, we found that PG synthesis by the symbionts decreases as they enter the slow-growing stage. Surprisingly, in contrast to the most mature crypts (i.e., Crypt 1) of juvenile animals, most of the symbiont cells in the least mature crypts (i.e., Crypt 3) were not expelled and, instead, remained in the slow-growing state throughout the day, with almost no cell division. Consistent with this observation, the expression of the gene encoding the PG-remodeling enzyme, L,D-transpeptidase (LdtA), was greatest during the slowly growing stage of Crypt 1 but, in contrast, remained continuously high in Crypt 3. Finally, deletion of the ldtA gene resulted in a symbiont that grew and survived normally in culture, but was increasingly defective in competing against its parent strain in the crypts. This result suggests that remodeling of the PG to generate additional 3–3 linkages contributes to the bacterium’s fitness in the symbiosis, possibly in response to stresses encountered during the very slow-growing stage.

• The role of substrate-binding interactions in regulating the formation and function of biomolecular condensates is under-explored.
• Studying BET family transcription factor BRD4, the authors performed coarse-grained simulations and live cell experiments to elucidate the kinetic and thermodynamic properties of condensation that are affected by acetylated chromatin binding. They found that acetylated chromatin binding targets and enhances BRD4 condensation.
• This provides a molecular and physical explanation of the relationship between nuclear condensates and epigenetically modified chromatin that results in their mutual spatiotemporal regulation, suggesting that epigenetic modulation is an important mechanism by which transcriptional condensates target specific chromatin loci.

Nuclear compartments form via biomolecular phase separation, mediated through multivalent properties of biomolecules concentrated within condensates. Certain compartments are associated with specific chromatin regions, including transcriptional initiation condensates, which are composed of transcription factors and transcriptional machinery, and form at acetylated regions including enhancer and promoter loci. While protein self-interactions, especially within low-complexity and intrinsically disordered regions, are known to mediate condensation, the role of substrate-binding interactions in regulating the formation and function of biomolecular condensates is underexplored. Here, utilizing live-cell experiments in parallel with coarse-grained simulations, we investigate how chromatin interaction of the transcriptional activator BRD4 modulates its condensate formation. We find that both kinetic and thermodynamic properties of BRD4 condensation are affected by chromatin binding: nucleation rate is sensitive to BRD4–chromatin interactions, providing an explanation for the selective formation of BRD4 condensates at acetylated chromatin regions, and thermodynamically, multivalent acetylated chromatin sites provide a platform for BRD4 clustering below the concentration required for off-chromatin condensation. This provides a molecular and physical explanation of the relationship between nuclear condensates and epigenetically modified chromatin that results in their mutual spatiotemporal regulation, suggesting that epigenetic modulation is an important mechanism by which the cell targets transcriptional condensates to specific chromatin loci.

• Microtubules rely on dynamics for their critical functions. In plant cells, the impact of damage and repair on microtubule dynamics has not been determined, due to the lack of tools for visualizing microtubule damage–repair events.
• The authors established a method for visualizing damage–repair sites on microtubule lattices and used this to reveal that cortical microtubules undergo frequent damage–repair in plant cells and the damage–repair leads to instability of microtubules.
• This work presents new evidence of microtubule damage–repair in plant cells and provides valuable tools for further research in the field.

Microtubules rely on dynamic assembly and disassembly for their functions. Increasing evidences support that the damage–repair of microtubule lattices can affect microtubule dynamics in vitro and in animal cells. Here we successfully established a way for visualizing damage–repair sites on microtubule lattices in plant cells, via labeling the tubulin proteins with the photoconvertible fluorescent protein mEOS3.2. We observed that the crossovers of the microtubule lattice were more prone to be damaged and repaired, with the frequency of damage–repair events positively correlated with the crossing angle between microtubules. The microtubules with damage–repair events displayed shorter lifespans and significantly increased severing frequency compared with the undamaged microtubules. These observations suggested that the damage–repair events promoted instability of cortical microtubules in plant cells.

• It has not been possible to globally inhibit microtubule (MT) polymerization into dendritic spines without affecting MT dynamics throughout the entire neuron as well as any neighboring cells
• Transfection with the LifeAct-MTED construct inhibits MT polymerization into dendritic spines in a spatially and temporally specific manor, without affecting native MT dynamics throughout the dendritic shaft
• The LifeAct-MTED construct will allow for elucidation of the function of MT entry into dendritic spines as well as other actin-MT interactions in both neuronal and nonneuronal cells.

Dendritic spines, the mushroom-shaped extensions along dendritic shafts of excitatory neurons, are critical for synaptic function and are one of the first neuronal structures disrupted in neurodevelopmental and neurodegenerative diseases. Microtubule (MT) polymerization into dendritic spines is an activity-dependent process capable of affecting spine shape and function. Studies have shown that MT polymerization into spines occurs specifically in spines undergoing plastic changes. However, discerning the function of MT invasion of dendritic spines requires the specific inhibition of MT polymerization into spines, while leaving MT dynamics in the dendritic shaft, synaptically connected axons and associated glial cells intact. This is not possible with the unrestricted, bath application of pharmacological compounds. To specifically disrupt MT entry into spines we coupled a MT elimination domain (MTED) from the Efa6 protein to the actin filament-binding peptide LifeAct. LifeAct was chosen because actin filaments are highly concentrated in spines and are necessary for MT invasions. Temporally controlled expression of this LifeAct-MTED construct inhibits MT entry into dendritic spines, while preserving typical MT dynamics in the dendrite shaft. Expression of this construct will allow for the determination of the function of MT invasion of spines and more broadly, to discern how MT-actin interactions affect cellular processes.

• Dendritic polarity is maintained by the arrest of dendrite-selective vesicles in the proximal axon, but the mechanism that mediates this arrest is unknown.
• The authors define a new molecular model of kinesin-3 family member KIF13A regulation by MARK2-mediated phosphorylation in the proximal axon. KIF13A is then recognized by 14-3-3 family members, resulting in kinesin-vesicle dissociation and termination of anterograde transport.
• These results indicate that regulation of kinesin binding to dendritic vesicles, rather than vesicle capture, is the key regulatory event that terminates anterograde transport of dendrite-selective vesicles at the base of the axon.

Neurons are polarized and typically extend multiple dendrites and one axon. To maintain polarity, vesicles carrying dendritic proteins are arrested upon entering the axon. To determine whether kinesin regulation is required for terminating anterograde axonal transport, we overexpressed the dendrite-selective kinesin KIF13A. This caused mistargeting of dendrite-selective vesicles to the axon and a loss of dendritic polarity. Polarity was not disrupted if the kinase MARK2/Par1b was coexpressed. MARK2/Par1b is concentrated in the proximal axon, where it maintains dendritic polarity–likely by phosphorylating S1371 of KIF13A, which lies in a canonical 14-3-3 binding motif. We probed for interactions of KIF13A with 14-3-3 isoforms and found that 14-3-3β and 14-3-3ζ bound KIF13A. Disruption of MARK2 or 14-3-3 activity by small molecule inhibitors caused a loss of dendritic polarity. These data show that kinesin regulation is integral for dendrite-selective transport. We propose a new model in which KIF13A that moves dendrite-selective vesicles in the proximal axon is phosphorylated by MARK2. Phosphorylated KIF13A is then recognized by 14-3-3, which causes dissociation of KIF13A from the vesicle and termination of transport. These findings define a new paradigm for the regulation of vesicle transport by localized kinesin tail phosphorylation, to restrict dendrite-selective vesicles from entering the axon.

• The chromosome-microtubule coupling machinery, the KMN (Knl1/Mis12/Ndc80) network, has a cell division-independent role in establishing the proper structure of C. elegans sensory nervous system, but the underlying mechanism is not known.
• The authors show that the KMN network is required for correct placement of axons within the C. elegans nerve ring. Structure-function analysis shows that neuronal KMN function requires both its signaling and microtubule-binding activities.
• These findings suggest that the communication and coordination between the KMN microtubule-binding and signaling machinery might be key to axon organization, offering new avenues to investigate the mechanisms involved in the organization of the nervous system.

The KMN (Knl1/Mis12/Ndc80) network at the kinetochore, primarily known for its role in chromosome segregation, has been shown to be repurposed during neurodevelopment. Here, we investigate the underlying neuronal mechanism and show that the KMN network promotes the proper axonal organization within the C. elegans head nervous system. Postmitotic degradation of KNL-1, which acts as a scaffold for signaling and has microtubule-binding activities at the kinetochore, led to disorganized ganglia and aberrant placement and organization of axons in the nerve ring - an interconnected axonal network. Through gene-replacement approaches, we demonstrate that the signaling motifs within KNL-1, responsible for recruiting protein phosphatase 1, and activating the spindle assembly checkpoint are required for neurodevelopment. Interestingly, while the microtubule-binding activity is crucial to KMN’s neuronal function, microtubule dynamics and organization were unaffected in the absence of KNL-1. Instead, the NDC-80 microtubule-binding mutant displayed notable defects in axon bundling during nerve ring formation, indicating its role in facilitating axon-axon contacts. Overall, these findings provide evidence for a noncanonical role for the KMN network in shaping the structure and connectivity of the nervous system in C. elegans during brain development.

• Based on their elegant experiments, concluded that the G2/M transition in fission yeast is controlled by a bistable switch, but we question that conclusion.
• Using detailed stochastic simulations, we show that the coexisting low and high Cdk1 activity states observed by Patterson et al. can be reproduced by either an irreversible/bistable or a reversible/ultrasensitive switch, and our analysis shows why the experiments of Patterson et al. are inconclusive.
• We suggest a decisive experiment to distinguish unequivocally between an irreversible/bistable and a reversible/ultrasensitive switch.

In favorable conditions, eukaryotic cells proceed irreversibly through the cell division cycle (G1-S-G2-M) in order to produce two daughter cells with the same number and identity of chromosomes of their progenitor. The integrity of this process is maintained by “checkpoints” that hold a cell at particular transition points of the cycle until all requisite events are completed. The crucial functions of these checkpoints seem to depend on irreversible bistability of the underlying checkpoint control systems. Bistability of cell cycle transitions has been confirmed experimentally in frog egg extracts, budding yeast cells and mammalian cells. For fission yeast cells, a recent paper by provides experimental evidence for an abrupt transition from G2 phase into mitosis, and we show that these data are consistent with a stochastic model of a bistable switch governing the G2/M checkpoint. Interestingly, our model suggests that their experimental data could also be explained by a reversible/sigmoidal switch, and stochastic simulations confirm this supposition. We propose a simple modification of their experimental protocol that could provide convincing evidence for (or against) bistability of the G2/M transition in fission yeast.

The endoplasmic reticulum (ER) is a single-copy organelle that cannot be generated de novo, suggesting coordination between the mechanisms overseeing ER integrity and those controlling the cell cycle to maintain organelle inheritance. The Unfolded Protein Response (UPR) is a conserved signaling network that regulates ER homeostasis. Here, we show that pharmacological and genetic inhibition of the UPR sensors IRE1, ATF6, and PERK in unstressed cells delays the cell cycle, with PERK inhibition showing the most penetrant effect, which was associated with a slowdown of the G₁-to-S/G₂ transition. Treatment with the small molecule ISRIB to bypass the effects of PERK-dependent phosphorylation of the translation initiation factor eIF2α had no such effect, suggesting that cell cycle timing depends on PERK’s kinase activity but is independent of eIF2α phosphorylation. Using complementary light and electron microscopy and flow cytometry-based analyses, we also demonstrate that the ER enlarges before mitosis. Together, our results suggest coordination between UPR signaling and the cell cycle to maintain ER physiology during cell division.

• The endoplasmic reticulum (ER) is a single-copy organelle that cannot be generated de novo, and its functions are controlled by the Unfolded Protein Response (UPR). How the UPR impacts the cell cycle remained unclear.
• By investigating this connection, the authors describe distinct roles for each UPR sensor, with PERK activity significantly affecting cell cycle beyond its typical function in the Integrated Stress Response (ISR). They also show that the ER expands before mitosis, potentially linking ER expansion to partitioning during mitosis.
• These findings highlight an unexplored UPR function in cell cycle regulation, potentially serving as a critical checkpoint for mitosis.

• It has been a mystery how lens epithelial cells differentiate into myofibroblasts in the FGF-rich, anti-fibrotic environment of the posterior lens capsule to cause secondary cataract, the most common vision disrupting complication of cataract surgery.
• Using a serum-free, primary lens epithelial cell culture system, the authors showed that FGF induced pT669-mediated desensitization of ErbB1 (EGFR) that was overcome by the previously reported increase in ErbB1 levels induced by TGFβ.
• This work demonstrates how TGFβ modulates ErbB signaling to induce epithelial to mesenchymal transition in the presence of otherwise lens fiber-eliciting levels of FGF and raises new strategies to prevent secondary cataract.

To cause vision-disrupting fibrotic secondary cataract (PCO), lens epithelial cells that survive cataract surgery must migrate to the posterior of the lens capsule and differentiate into myofibroblasts. During this process, the cells become exposed to the FGF that diffuses out of the vitreous body. In normal development, such relatively high levels of FGF induce lens epithelial cells to differentiate into lens fiber cells. It has been a mystery as to how lens cells could instead undergo a mutually exclusive cell fate, namely epithelial to myofibroblast transition, in the FGF-rich environment of the posterior capsule. We and others have reported that the ability of TGFβ to induce lens cell fibrosis requires the activity of endogenous ErbBs. We show here that lens fiber-promoting levels of FGF induce desensitization of ErbB1 (EGFR) that involves its phosphorylation on threonine 669 mediated by both ERK and p38 activity. Transinhibition of ErbB1 by FGF is overcome by a time-dependent increase in ErbB1 levels induced by TGFβ, the activation of which is increased after cataract surgery. Our studies provide a rationale for why TGFβ upregulates ErbB1 in lens cells and further support the receptor as a therapeutic target for PCO.

• The tissue-specific functions of mammalian actin nucleation factors are not well understood.
• Mice lacking the actin nucleation factor WHAMM show signs of a kidney disease resembling Fanconi Syndrome. Kidneys from WHAMM-deficient male mice display abnormalities in structure and reabsorption function. Cultured proximal tubule cells exhibit defects in autophagosome closure and autophagic cargo sequestration.
• WHAMM and the Arp2/3 complex are important for nutrient reabsorption in the kidney and autophagosome remodeling in proximal tubule cells.

The actin cytoskeleton is essential for many functions of eukaryotic cells, but the factors that nucleate actin assembly are not well understood at the organismal level or in the context of disease. To explore the function of the actin nucleation factor WHAMM in mice, we examined how Whamm inactivation impacts kidney physiology and cellular proteostasis. We show that male WHAMM knockout mice excrete elevated levels of albumin, glucose, phosphate, and amino acids, and display structural abnormalities of the kidney proximal tubule, suggesting that WHAMM activity is important for nutrient reabsorption. In kidney tissue, the loss of WHAMM results in the accumulation of the lipidated autophagosomal membrane protein LC3, indicating an alteration in autophagy. In mouse fibroblasts and human proximal tubule cells, WHAMM and its binding partner the Arp2/3 complex control autophagic membrane closure and cargo receptor recruitment. These results reveal a role for WHAMM-mediated actin assembly in maintaining kidney function and promoting proper autophagosome membrane remodeling.

• Solo, a RhoGEF, is involved in mechanical stress response; however, its molecular mechanism remains unclear.
• We revealed that Solo directly regulates the localization and activity of PDZ-RhoGEF, both RhoA-targeting for actin cytoskeletal remodeling. This signaling cascade is required to activate the cellular response to substrate stiffness.
• These findings can potentially lead to the discovery of novel molecular mechanisms that regulate the mechanical stress responses of cells via Solo.

Recent findings indicate that Solo, a RhoGEF, is involved in cellular mechanical stress responses. However, the mechanism of actin cytoskeletal remodeling via Solo remains unclear. Therefore, this study aimed to identify Solo-interacting proteins using the BioID, a proximal-dependent labeling method, and elucidate the molecular mechanisms of function of Solo. We identified PDZ-RhoGEF (PRG) as a Solo-interacting protein. PRG colocalized with Solo in the basal area of cells, depending on Solo localization, and enhanced actin polymerization at the Solo accumulation sites. Additionally, Solo and PRG interaction was necessary for actin cytoskeletal remodeling. Furthermore, the purified Solo itself had little or negligible GEF activity, even its GEF-inactive mutant directly activated the GEF activity of PRG through interaction. Moreover, overexpression of the Solo and PRG binding domains, respectively, had a dominant-negative effect on actin polymerization and actin stress fiber formation in response to substrate stiffness. Therefore, Solo restricts the localization of PRG and regulates actin cytoskeletal remodeling in synergy with PRG in response to the surrounding mechanical environment.

• Formins and their regulators have been studied for many years in yeast, but almost exclusively in mitotically dividing cells, leaving their roles in the pheromone response poorly understood.
• The authors here find that proper shmoo formation requires the formin Bni1 but not Bnr1, as well as the actin nucleation-promoting activities of Bud6, and a novel activity of Bil2 in spatially organizing Bni1 at mating projection tips.
• This work demonstrates the physiological importance of formin spatial regulation in shaping actin networks, which in turn shapes cell morphology, and establishes an ideal in vivo system for dissecting Bni1 regulation.

In response to pheromone Saccharomyces cerevisiae extend a mating projection. This process depends on the formation of polarized actin cables which direct secretion to the mating tip and translocate the nucleus for karyogamy. Here, we demonstrate that proper mating projection formation requires the formin Bni1, as well as the actin nucleation promoting activities of Bud6, but not the formin Bnr1. Further, Bni1 is required for pheromone gradient tracking. Our work also reveals unexpected new functions for Bil2 in the pheromone response. Previously we identified Bil2 as a direct inhibitor of Bnr1 during vegetative cell growth. Here, we show that Bil2 has Bnr1-independent functions in spatially focusing Bni1-GFP at mating projection tips, and in vitro Bil2 and its binding partner Bud6 organize Bni1 into clusters that nucleate actin assembly. bil2∆ cells also display entangled Bni1-generated actin cable arrays and defects in secretory vesicle transport and nuclear positioning. At low pheromone concentrations, bil2∆ cells are delayed in establishing a polarity axis, and at high concentrations they prematurely form a second and a third mating projection. Together, these results suggest that Bil2 promotes the proper formation and timing of mating projections by organizing Bni1 and maintaining a persistent axis of polarized growth.

• Stathmins are conserved across most animals and implicated in disease biology, but their evolutionary origin and cellular functions are unclear, especially in the case of STMND1, which has not been the subject of any cellular analysis.
• Our phylogenetic analysis argues that STMND1 is the human representative of the most ancient clade. STMND1 is primarily expressed in mutlicilliated epithelial cells, where it localizes to cilia.
• Our results indicate that tubulin binding negatively regulates the nuclear transport of STMND1, implying that tubulin binding may also regulate the localization or function of other membrane-bound stathmins.

Stathmins are small, unstructured proteins that bind tubulin dimers and are implicated in several human diseases, but whose function remains unknown. We characterized a new stathmin, STMND1 (Stathmin Domain Containing 1) as the human representative of an ancient subfamily. STMND1 features a N-terminal myristoylated and palmitoylated motif which directs it to membranes and a tubulin-binding stathmin-like domain (SLD) that contains an internal nuclear localization signal. Biochemistry and proximity labeling showed that STMND1 binds tubulin, and live imaging showed that tubulin binding inhibits translocation from cellular membranes to the nucleus. STMND1 is highly expressed in multiciliated epithelial cells, where it localizes to motile cilia. Overexpression in a model system increased the length of primary cilia. Our study suggests that the most ancient stathmins have cilium-related functions that involve sensing soluble tubulin.

• The kinesin-12 (KIF15) can substitute for kinesin-5 (Eg5) during spindle assembly, but KIF15 is less efficient in this role due to its reduced ability to generate microtubule sliding forces. It was hypothesized that microtubule-bundling factors that stabilize antiparallel microtubule overlaps may facilitate KIF15 activity during spindle assembly.
• The authors showed that PRC1, an antiparallel microtubule crosslinking protein, is required for KIF15-dependent spindle assembly, and that its overexpression can promote cancer-cell resistance to K5Is.
• This work has implications for cancer therapies that operate by blocking spindle assembly.

The spindle is a bipolar microtubule-based machine that is crucial for accurate chromosome segregation. Spindle bipolarity is generated by Eg5 (a kinesin-5), a conserved motor that drives spindle assembly by localizing to and sliding apart antiparallel microtubules. In the presence of Eg5 inhibitors (K5Is), KIF15 (a kinesin-12) can promote spindle assembly, resulting in K5I-resistant cells (KIRCs). However, KIF15 is a less potent motor than Eg5, suggesting that other factors may contribute to spindle formation in KIRCs. Protein Regulator of Cytokinesis 1 (PRC1) preferentially bundles antiparallel microtubules, and we previously showed that PRC1 promotes KIF15-microtubule binding, leading us to hypothesize that PRC1 may enhance KIF15 activity in KIRCs. Here, we demonstrate that: 1) loss of PRC1 in KIRCs decreases spindle bipolarity, 2) overexpression of PRC1 increases spindle formation efficiency in KIRCs, 3) overexpression of PRC1 protects K5I naïve cells against the K5I S-trityl-L-cysteine (STLC), and 4) PRC1 overexpression promotes the establishment of K5I resistance. These effects are not fully reproduced by a TPX2, a microtubule bundler with no known preference for microtubule orientation. These results suggest a model wherein PRC1-mediated bundling of microtubules creates a more favorable microtubule architecture for KIF15-driven mitotic spindle assembly in the context of Eg5 inhibition.

• The assembly of BAR domain-containing endosomal coats such as retromer involves BAR–BAR pairing, though recent work suggests that additional structural features contribute to subunit selection
• We identified features in the unstructured N-termini of two paralogous yeast SNX–BARs, Vps5 and Vin1, that mediate the specific formation of the related retromer and VINE complexes
• Our findings highlight how disordered regions in SNX–BARs help determine the composition of sorting complexes, thereby clarifying mechanisms of modular membrane coat assembly

Endosomal coats incorporate membrane-binding subunits such as sorting nexin (SNX) proteins. The Saccharomyces cerevisiae SNX–BAR paralogs Vin1 and Vps5 are respective subunits of the endosomal VINE and retromer complexes whose dimerizing BAR domains are required for complex assembly and membrane association. However, a degree of promiscuity is predicted for yeast BAR–BAR pairings, and recent work has implicated the unstructured N-terminal domains of Vin1 and Vps5 in coat formation. Here, we map N-terminal signals in both SNX–BAR paralogs that contribute to the assembly and function of two distinct endosomal coats in vivo. Whereas Vin1 leverages a polybasic region and adjacent hydrophobic motif to bind Vrl1 and form VINE, the N-terminus of Vps5 interacts with the retromer subunit Vps29 at two sites, including a conserved hydrophobic pocket in Vps29 that engages other accessory proteins in humans. We also examined the sole isoform of Vps5 from the milk yeast Kluyveromyces lactis and found that ancestral yeasts may have used a nested N-terminal signal to form both VINE and retromer. Our results suggest that the specific assembly of Vps5-family SNX–BAR coats depends on inputs from unique N-terminal sequence features in addition to BAR domain coupling, expanding our understanding of endosomal coat biology.